Assay panels

ABSTRACT

Described herein are kits and components thereof used for a multiplexed analysis of a set of cytokines.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of copending application Ser. No. 14/146,066, filed on Jan. 2, 2014, which claims the benefit of U.S. Provisional Application No. 61/748,626, filed on Jan. 3, 2013, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This application relates to kits used for the detection of cytokines using electrochemiluminescent technology.

BACKGROUND OF THE INVENTION

Cytokines are the soluble factors that mediate acute and chronic inflammatory responses, and are involved in many physiological events from wound healing to autoimmune disorders. They are important regulators of cell-mediated and humoral immune responses and their differential expression has been associated with a wide array of immune disorders. They function on a variety of cell types, having stimulatory or inhibitory effects on proliferation, differentiation, and maturation. Therefore, measuring the level of only a single cytokine in any biological system provides only partial information relevant to the response on a systemic level. Comprehensive tests for cytokine levels generally aim to measure the concentrations of a large set of cytokines to gain a better understanding of the underlying physiology.

The enzyme-linked immunosorbent assay (ELISA) is the most commonly used and reported method for the quantitation of secreted cytokines. However, ELISA can only detect one analyte per reaction in individual assay wells. This leads to high reagent cost, excessive technician time, and the need to use large sample volumes to generate each results. The ability to detect and quantitate many cytokines simultaneously in the same sample via a robust multiplexed assay would reduce costs and improve efficiency. The advantages of multiplex technology over conventional assay methods include simultaneous analyte detection, reduced reagent handling, high assay throughput, and decreased sample and reagent volumes.

SUMMARY OF THE INVENTION

The invention provides a kit for the analysis of a cytokine panel comprising:

i. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: IFN-gamma, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNFalpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

ii. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

iii. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following human analytes are bound: Eotaxin, MIP-1 alpha, Eotaxin-3, TARO, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins;

iv. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following rat analytes are bound: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins; or

v. (a) a multi-well assay plate comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to the following mouse analytes are bound: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha; (b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said human analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

Also provided is a method of manufacturing a kit or a lot of kit such as those described herein that includes: (a) subjecting a preliminary set of detection antibodies specific for said human analytes to CIEF, DLS, and Experion; (b) selecting qualified detection antibodies from said preliminary set of detection antibodies based on said CIEF, DLS, and Experion testing; (c) subjecting a preliminary set of capture antibodies specific for said human analytes to CIEF, DLS, and Experion; and (b) selecting qualified capture antibodies from said preliminary set of capture antibodies based on said CIEF, DLS, and Experion testing. In a preferred embodiment, a lot of kits is manufacturing using this protocol and meets one or more of the following specifications: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.

In a preferred embodiment, the invention provides a kit for the analysis of two or more cytokine panels comprising: (a) two or more multi-well assay plates each comprising a plurality of wells, each well comprising ten discrete binding domains to which capture antibodies to a set of analytes are bound, wherein said set of analytes is selected from the group consisting of:

(i) human analytes: IFN-gamma, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha;

(ii) human analytes: GM-CSF, IL-1 alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A;

(iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4;

(iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or

(v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha;

(b) in one or more vials, containers, or compartments, a set of labeled detection antibodies specific for said analytes; and (c) in one or more vials, containers, or compartments, a set of calibrator proteins.

An additional embodiment of the invention is a 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, an x- and y-axis of the plate top and bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°, wherein (a) Δx is the difference between a center of the spot pattern and a center of a well along the x axis of the plate; (b) Δy is the difference between the center of a spot pattern and a center of the well along the y axis of the plate; and (c) α is a counter-clockwise angle between the x axis of the plate bottom and the x axis of the plate top.

Moreover, the invention contemplates a 10-spot 96-well multi-well plate, wherein each plate comprises a plate top, a plate bottom, and each well comprises a spot pattern, wherein the plate meets the following specifications: (a) a length range of 3.8904-3.9004 inches; (b) a width range of 2.4736-2.4836 inches; and (c) well to well spacing of 0.3513-0.3573 inches.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(a)-(c) illustrate a 10-spot pattern in a well of a multi-well plate (panel (a)), its placement in a 96-well 10-spot plate (panel (b)), and the principles of an immunoassay conducted using a multi-well assay plate such as those described herein.

FIGS. 2(a)-(e) are standard curves for each of the five cytokine panels.

FIGS. 3(a)-(b) shows the configuration of a 96 well multi-well assay plate.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein, the term “sample” is intended to mean any biological fluid, cell, tissue, organ or combinations or portions thereof, which includes or potentially includes a biomarker of a disease of interest. For example, a sample can be a histologic section of a specimen obtained by biopsy, or cells that are placed in or adapted to tissue culture. A sample further can be a subcellular fraction or extract, or a crude or substantially pure nucleic acid molecule or protein preparation. In one embodiment, the samples that are analyzed in the assays of the present invention are blood, peripheral blood mononuclear cells (PBMC), isolated blood cells, serum and plasma. Other suitable samples include biopsy tissue, intestinal mucosa, saliva, cerebral spinal fluid, and urine.

The present invention relates to a kit for the analysis of a cytokine panel. At least five assay panels are contemplated and each kit is configured to analyze one of the following panels:

TABLE 1 Cytokine Assay Panels Panel Species Analytes 1 Human IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-13, TNFalpha 2 Human GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, VEGF-A 3 Human Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, MCP-4 4 Rat IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, TNF-alpha 5 Mouse IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, TNF-alpha

The kits can include (a) a single panel arrayed on a multi-well plate which is configured to be used in an electrochemiluminescence assay, as well as (b) associated consumables, e.g., detection antibodies, calibrators, and optional diluents and/or buffers. Alternatively, the multi-well plates and associated consumables can be provided separately. Still further, a kit can include two or more multi-well plates with panels arrayed thereon, i.e., panels 1-5, and the associated consumables can be provided in the kit or separately.

Panels 1, 2, 4, and 5 include inflammation-related and/or growth factor biomarkers that are important for inflammation response, immunity, and regulation of numerous biological processes. These secreted biomarkers can be detected in a variety of tissues and bodily fluids and their over- or under-expression can indicate a shift in biological equilibrium of the body. These panels also consist of many of the Th1/Th2 pathway biomarkers. The biomarkers in these panels are involved in numerous disorders such as rheumatoid arthritis, Alzheimer's disease, asthma, atherosclerosis, allergies, systematic lupus erythematosus, obesity, cancer, depression, multiple sclerosis, diabetes, psoriasis, and Crohn's disease, among others.

Panel 3 consists of eight CC chemokine assays (MCP-1, MIP-1a, MIP-1b, Eotaxin, MCP-4, TARC, MDC, and Eotaxin-3) and two CXC chemokine assays (IL-8 and IP-10). Chemokines are small chemotactic cytokines with molecular weights around 8-10 kDa that are capable of inducing directed chemotaxis. The four cysteine residues in conserved locations result in their compact 3-dimensional structure. Based on the spacing of the first two cysteine residues, they are divided into four families of chemokines—CC chemokines, CXC chemokines, C chemokines, and CX3C chemokines, where C represents cysteine and X represents any other amino acids. Chemokines function by activating specific G protein-coupled receptors resulting in migration of inflammatory and non-inflammatory cells. The pro-inflammatory chemokines are responsible for migration of immune cells to the infection site while the homeostatic chemokines are responsible for the migration of cells for the purpose of tissue maintenance and development. Chemokines are associated with number of diseases.

Panels 1-5 are configured in a multi-well assay plate including a plurality of wells, each well having an array with 10 “spots” or discrete binding domains. An example of a 10-spot well is shown in FIG. 1(a) and the incorporation of that well into a multi-well plate is shown in FIG. 1(b). A capture antibody to each analyte is immobilized on a binding domain in the well and that capture antibody is used to detect the presence of the target analyte in an immunoassay as illustrated in FIG. 1(c). Briefly, a sample suspected of containing that analyte is added to the well and if present, the analyte binds to the capture antibody at the designated binding domain. The presence bound analyte on the binding domain is detected by adding labeled detection antibody. The detection antibody also binds to the analyte forming a “sandwich” complex (capture antibody-analyte-detection antibody) on the binding domain. The location of each analyte in Panels 1-5 in this 10-spot pattern is identified in Table 2.

TABLE 2 Spot Pattern Configuration Per Panel Panel Species Spot Location Analytes 1 Human 1 IFN-gamma 2 IL-1beta 3 IL-2 4 IL-4 5 IL-6 6 IL-8 7 IL-10 8 IL-12p70 9 IL-13 10 TNFalpha 2 Human 1 GM-CSF 2 IL-1alpha 3 IL-5 4 IL-7 5 IL-12/IL-23 p40 6 IL-15 7 IL-16 8 IL-17A 9 TNF-beta 10 VEGF-A 3 Human 1 Eotaxin, 2 MIP-1 alpha, 3 Eotaxin-3 4 TARC 5 IP-10 8 MIP-1 beta 7 IL-8 8 MCP-1 9 MDC 10 MCP-4 4 Rat 1 IFN-gamma 2 IL-2 3 IL-4 4 IL-1 beta 5 IL-5 8 IL-6 7 KC/GRO 8 IL-10 9 IL-13 10 TNF-alpha 5 Mouse 1 IFN-gamma 2 IL-1-beta 3 IL-2 4 IL-4 5 IL-5 6 IL-6 7 KC/GRO 8 IL-10 9 IL-12p70 10 TNF-alpha

The multiplexed immunoassay kits described herein allow a user to simultaneously quantify multiple biomarkers. The panels are selected and optimized such that the individual assays function well together. The sample may require dilution prior to being assayed. Sample dilutions for specific sample matrices of interest are optimized for a given panel to minimize sample matrix effects and to maximize the likelihood that all the analytes in the panel will be within the dynamic range of the assay. In a preferred embodiment, all of the analytes in the panel are analyzed with the same sample dilution in at least one sample type. In another preferred embodiment, all of the analytes in a panel are measured using the same dilution for most sample types.

For a given panel, the detection antibody concentration and the number of labels per protein (L/P ratio) for the detection antibody are adjusted to bring the expected levels of all analytes into a quantifiable range at the same sample dilution. If one wants to increase the high end of the quantifiable range for a given analyte, then the L/P can be decreased and/or the detection antibody concentration is decreased. On the other hand, if one wants to increase the lower end of the quantifiable range, the L/P can be increased, the detection antibody concentration can be increased if it is not at the saturation level, and/or the background signal can be lowered.

Calibration standards for use with the assay panels are selected to provide the appropriate quantifiable range with the recommended sample dilution for the panel. The calibration standards have known concentrations of one of more of the analytes in the panel. Concentrations of the analytes in unknown samples are determined by comparison to these standards. In one embodiment, calibration standards comprise mixtures of the different analytes measured by an assay panel. Preferably, the analyte levels in a combined calibrator are selected such that the assay signals for each analyte are comparable, e.g., within a factor of two, a factor of five or a factor of 10. In another embodiment, calibration standards include mixtures of analytes from multiple different assay panels.

A calibration curve may be fit to the assay signals measured with calibration standards using, e.g., curve fits known in the art such as linear fits, 4-parameter logistic (4-PL) and 5-parameter (5-PL) fits. Using such fits, the concentration of analytes in an unknown sample may be determined by backfitting the measured assay signals to the calculated fits. Measurements with calibration standards may also be used to determine assay characteristics such as the limit of detection (LOD), limit of quantification (LOQ), dynamic range, and limit of linearity (LOL).

A kit includes the following assay components: a multi-well assay plate configured to conduct an immunoassay for one of the panels described herein, a set of detection antibodies for the analytes in the panel (wherein the set comprises individual detection antibodies and/or a composition comprising a blend of one or more individual detection antibodies), and a set of calibrators for the analytes in the panel (wherein the set comprises individual calibrator protein compositions and/or a composition comprising a blend of one or more individual calibrator proteins). The kit can also include one of more of the following additional components: a blocking buffer (used to block assay plates prior to addition of sample), an antibody diluent (used to dilute stock detection antibody concentrations to the working concentration), an assay diluent (used to dilute samples), a calibrator diluent (used to dilute or reconstitute calibration standards) and a read buffer (used to provide the appropriate environment for detection of assay labels, e.g., by an ECL measurement). The antibody and assay diluents are selected to reduce background, optimize specific signal, and reduce assay interference and matrix effect. The calibrator diluent is optimized to yield the longest shelf life and retention of calibrator activity. The blocking buffer should be optimized to reduce background. The read buffer is selected to yield the appropriate sensitivity, quantifiable range, and slowest off-rate. The reagent components of the kit can be provided as liquid reagents, lyophilized, or combinations thereof, diluted or undiluted, and the kit includes instructions for appropriate preparation of reagents prior to use. In a preferred embodiment, a set of detection antibodies are included in the kit comprising a plurality of individual detection antibody compositions in liquid form. Moreover, the set of calibrators provided in the kit preferably comprise a lyophilized blend of calibrator proteins. Still further, the kit includes a multi-well assay plate that has been pre-coated with capture antibodies and exposed to a stabilizing treatment to ensure the integrity and stability of the immobilized antibodies.

As part of a multiplexed panel development, assays are optimized to reduce calibrator and detection antibody non-specific binding. In sandwich immunoassays, specificity mainly comes from capture antibody binding. Some considerations for evaluating multiplexed panels include: (a) detection antibody non-specific binding to capture antibodies is reduced to lower background of assays in the panel, and this can be achieved by adjusting the concentrations and UP of the detection antibodies; (b) non-specific binding of detection antibodies to other calibrators in the panel is also undesirable and should be minimized; (c) non-specific binding of other calibrators in the panel and other related analytes should be minimized; if there is calibrator non-specific binding, it can reduce the overall specificity of the assays in the panel and it can also yield unreliable results as there will be calibrator competition to bind the capture antibody.

Different assays in the panel may require different incubation times and sample handling requirements for optimal performance. Therefore, the goal is to select a protocol that's optimized for most assays in the panel. Optimization of the assay protocol includes, but is not limited to, adjusting one or more of the following protocol parameters: timing (incubation time of each step), preparation procedure (calibrators, samples, controls, etc.), and number of wash steps.

The reagents used in the kits, e.g., the detection and capture antibodies and calibrator proteins, are preferably subjected to analytical testing and meet or exceed the specifications for those tests. The analytical tests that can be used to characterize kit materials include but are not limited to, CIEF, DLS, reducing and/or non-reducing EXPERION, denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof. In a preferred embodiment, the materials are characterized by CIEF, DLS, and reducing and non-reducing EXPERION. One or more additional tests, including but not limited to denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof, can also be used to characterize the materials. In a preferred embodiment, the materials are also subjected to functional testing, i.e., a binding assay for the target analyte, as well as one or more characterization tests, such as those listed above. If the materials do not meet or exceed the specifications for the functional and/or characterization tests, they can be subjected to additional purification steps and re-tested. Each of these tests and the metrics applied to the analysis of raw materials subjected to these tests are described below:

Capillary Isoelectric Focusing (CIEF) is a technique commonly used to separate peptides and proteins, and it is useful in the detection of aggregates. During a CIEF separation, a capillary is filled with the sample in solution and when voltage is applied, the ions migrate to a region where they become neutral (pH=pI). The anodic end of the capillary sits in acidic solution (low pH), while the cathodic end sits in basic solution (high pH). Compounds of equal isoelectric points (pI) are “focused” into sharp segments and remain in their specific zone, which allows for their distinct detection based on molecular charge and isoelectric point. Each specific antibody solution will have a fingerprint CIEF that can change over time. When a protein solution deteriorates, the nature of the protein and the charge distribution can change. Therefore, CIEF is a particularly useful tool to assess the relative purity of a protein solution and it is a preferred method of characterizing the antibodies and calibrators in the plates and kits described herein. The metrics used in CIEF include pI of the main peak, the pI range of the solution, and the profile shape, and each of these measurements are compared to that of a reference standard.

Dynamic Light Scattering (DLS) is used to probe the diffusion of particulate materials either in solution or in suspension. By determining the rate of diffusion (the diffusion coefficient), information regarding the size of particles, the conformation of macromolecular chains, various interactions among the constituents in the solution or suspension, and even the kinetics of the scatterers can be obtained without the need for calibration. In a DLS experiment, the fluctuations (temporal variation, typically in a μs to ms time scale) of the scattered light from scatterers in a medium are recorded and analyzed in correlation delay time domain. Like CIEF, each protein solution will generate a fingerprint DLS for the particle size and it's ideally suited to detect aggregation. All IgGs, regardless of binding specificity, will exhibit the same DLS particle size. The metrics used to analyze a protein solution using DLS include percentage polydispersity, percentage intensity, percentage mass, and the radius of the protein peak. In a preferred embodiment, an antibody solution meets or exceeds one or more of the following DLS specifications: (a) radius of the antibody peak: 4-8 nm (antibody molecule size); (b) polydispersity of the antibody peak: <40% (measure of size heterogeneity of antibody molecules): (c) intensity of the antibody peak: >50% (if other peaks are present, then the antibody peak is the predominant peak); and (d) mass in the antibody peak: >50%.

Reducing and non-reducing gel electrophoresis are techniques well known in the art. The EXPERION™ (Bio-Rad Laboratories, Inc., www.bio-rad.com) automated electrophoresis station performs all of the steps of gel-based electrophoresis in one unit by automating and combining electrophoresis, staining, destaining, band detection, and imaging into a single step. It can be used to measure purity. Preferably, an antibody preparation is greater 50% pure by Experion, more preferably, greater than 75% pure, and most preferably greater than 80% pure. Metrics that are applied to protein analysis using non-reducing Experion include percentage total mass of protein, and for reducing Experion they include percentage total mass of the heavy and light chains in an antibody solution, and the heavy to light chain ratio.

Multi-Angle Light Scattering (MALS) detection can be used in the stand-alone (batch) mode to measure specific or non-specific protein interactions, as well as in conjunction with a separation system such as flow field flow fractionation (FFF) or size exclusion chromatography (SEC). The combined SEC-MALS method has many applications, such as the confirmation of the oligomeric state of a protein, quantification of protein aggregation, and determination of protein conjugate stoichiometry. Preferably, this method is used to detect molecular weight of the components of a sample.

In a preferred embodiment, an assay is conducted in a single assay chamber, such as a single well of an assay plate or an assay chamber that is an assay chamber of a cartridge. In a preferred embodiment, the kits of the invention include multi-well assay plates that are configured to conduct an electrochemiluminescence measurement as described for example, in US 20040022677; US 20050052646; US 20050142033; US 20040189311, each of which is incorporated herein by reference in their entireties. Assay plates and plate readers are now commercially available (MULTI-SPOT® and MULTI-ARRAY® plates and SECTOR® instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics, LLC, Gaithersburg, Md.).

As used herein, a lot of kits comprise a group of kits comprising kit components that meet a set of kit release specifications. A lot can include at least 10, at least 100, at least 500, at least 1,000, at least 5,000, or at least 10,000 kits and a subset of kits from that lot are subjected to analytical testing to ensure that the lot meets or exceeds the release specifications. In one embodiment, the release specifications include but are not limited to kit processing, reagent stability, and kit component storage condition specifications. Kit processing specifications include the maximum total sample incubation time and the maximum total time to complete an assay using the kit. Reagent stability specifications include the minimum stability of each reagent component of the kit at a specified storage temperature. Kit storage condition specifications include the range of storage temperatures for all components of the kit, the maximum storage temperature for frozen components of the kit, and the maximum storage temperature for non-frozen components of the kit. A subset of kits in a lot are reviewed in relation to these specifications and the size of the subset depends on the lot size. In a preferred embodiment, for a lot of up to 300 kits, a sampling of 4-7 kits are tested; for a lot of 300-950 kits, a sampling of 8-10 kits are tested; and for a lot of greater than 950 kits, a sampling of 10-12 kits are tested. Alternatively or additionally, a sampling of up to 1-5% preferably up to 1-3%, and most preferably up to 2% is tested.

In addition, each lot of multi-well assay plates is preferably subjected to uniformity and functional testing. A subset of plates in a lot are subjected to these testing methods and the size of the subset depends on the lot size. In a preferred embodiment, for a lot of up to 300 plates, a sampling of 4-7 plates are tested; for a lot of 300-950 plates, a sampling of 8-10 plates are tested; and for a lot of greater than 950 plates, a sampling of 10-12 plates are tested. Alternatively or additionally, a sampling of up to 1-5% preferably up to 1-3%, and most preferably up to 2% is tested. The uniformity and functional testing specifications are expressed in terms of % CV, Coefficient of Variability, which is a dimensionless number defined as the standard deviation of a set of measurements, in this case, the relative signal detected from binding domains across a plate, divided by the mean of the set.

One type of uniformity testing is protein A/G testing. Protein A/G binding is used to confirm that all binding domains within a plate are coupled to capture antibody. Protein A/G is a recombinant fusion protein that combines IgG binding domains of Protein A and protein G and it binds to all subclasses of human IgG, as well as IgA, IgE, IgM and, to a lesser extent, IgD. Protein A/G also binds to all subclasses of mouse IgG but not mouse IgA, IgM, or serum albumin, making it particularly well suited to detect mouse monoclonal IgG antibodies without interference from IgA, IgM, and serum albumin that might be present in the sample matrix. Protein A/G can be labeled with a detectable moiety, e.g., a fluorescent, chemiluminescent, or electrochemiluminescent label, preferably an ECL label, to facilitate detection. Therefore, if capture antibody is adhered to a binding domain of a well, it will bind to labeled protein A/G, and the relative amount of capture antibody bound to the surface across a plate can be measured.

In addition to the uniformity testing described above, a uniformity metric for a subset of plates within a lot can be calculated to assess within-plate trending. A uniformity metric is calculated using a matrix of normalized signals from protein A/G and/or other uniformity or functional tests. The raw signal data is smoothed by techniques known in the art, thereby subtracting noise from the raw data, and the uniformity metric is calculated by subtracting the minimum signal in the adjusted data set from the maximum signal.

In a preferred embodiment, a subset of plates in a lot is subjected to protein A/G and functional testing and that subset meet or exceed the following specifications:

TABLE 3(a) Plate Metrics Preferred Specification for a subset Metric of 96 well multi-well plates Average intraplate CV ≦10% Maximum intraplate CV ≦13% Average Uniformity ≦25% Maximum Uniformity ≦37% CV of intraplate averages ≦18% Signal, lower boundary >1500 Signal, upper boundary   <10⁽⁶⁾

As disclosed in U.S. Pat. No. 7,842,246 to Wohlstadter et al., the disclosure of which is incorporated herein by reference in its entirety, each plate consists of several elements, e.g., a plate top, a plate bottom, wells, working electrodes, counter electrodes, reference electrodes, dielectric materials, electrical connects, and assay reagents. The wells of the plate are defined by holes/openings in the plate top. The plate bottom can be affixed, manually or by automated means, to the plate top, and the plate bottom can serve as the bottom of the well. Plates may have any number of wells of any size or shape, arranged in any pattern or configuration, and they can be composed of a variety of different materials. Preferred embodiments of the invention use industry standard formats for the number, size, shape, and configuration of the plate and wells. Examples of standard formats include 96, 384, 1536, and 9600 well plates, with the wells configured in two-dimensional arrays. Other formats may include single well plates (preferably having a plurality of assay domains that form spot patterns within each well), 2 well plates, 6 well plates, 24 well plates, and 6144 well plates. Each well of the plate includes a spot pattern of varying density, ranging from one spot within a well to 2, 4, 7, 9, 10, 16, 25, etc. In a preferred embodiment, the plates used in the kits of the invention comprise 10-spot 96-well plates.

Each plate is assembled according to a set of preferred specifications. In a preferred embodiment, a plate bottom meets or exceeds the following specifications:

TABLE 3(b) Plate bottom specifications 96-well (round well) specifications in Parameter inches Length range (C to C)* 3.8904-3.9004 (A1-A12 and H1-H12)** Width range (C to C) 2.4736-2.4836 (A1-A12 and H1-H12) Well to well spacing 0.3513-0.3573 *C to C well distance is the center of spot to center of spot distance between the outermost wells of a plate. **As shown in FIG. 3, a 96-well multi-well plate includes a set of wells arranged in an 8 × 12 array, wherein the rows on the short side of the plate are identified by A-H, and the columns on the long side of the plate are identified by 1-12. Therefore, length and width can be measured in row A1-A12 and compared to that of row H1-H12.

In a further preferred embodiment, the plate also meets or exceeds defined specifications for alignment of a spot pattern within a well of the plate. These specifications include three parameters: (a) Δx, the difference between the center of the spot pattern and the center of the well along the x axis of the plate (column-wise, long axis); (b) Δy, the difference between the center of the spot pattern and the center of the well along the y axis of the plate (row-wise, short axis); and (c) α, the counter-clockwise angle between the long axis of the plate bottom and the long axis of the plate top of a 96-well plate. In a preferred embodiment, the plate meets or exceeds the following specifications: Δx≦0.2 mm. Δy≦0.2 mm, and α≦0.1°.

The following non-limiting examples serve to illustrate rather than limit the present invention.

EXAMPLES Example 1. Reagent Preparation

All reagents were brought to room temperature and diluents were thawed in water at room temperature.

(i) Preparation of Standards

Multi-analyte lyophilized calibrator blends and all diluents for each panel were obtained from Meso Scale Discovery (Rockville, Md.) which yield the recommended highest standard upon reconstitution in one mL of diluent. The lyophilized calibrator was reconstituted and kept on ice. Seven (7) standard solutions and a zero calibrator blank were prepared for up to 4 replicates as follows: (x) The highest standard was prepared by adding 1000 μL of diluent to the lyophilized calibrator vial. The solution was mixed by vortexing and keep on wet ice for a minimum of 5 minutes prior to use. (y) The next standard was prepared by transferring 75 μL of the highest standard to 225 μL of diluent. The solution was mixed well and the procedure repeated 4-fold serial dilutions 5 additional times to generate 7 standards. (z) Diluent was used as the blank. Once reconstituted to the recommended highest standard in Diluent 2, the multi-analyte lyophilized calibrator for each kit is stable at 2-8° C. for 30 days.

(ii) Sample Collection & Handling

When preparing serum, samples were allowed to clot for two hours at room temperature. Plasma prepared in heparin tubes commonly display additional clotting following thawing of the sample. Both serum and plasma were centrifuged for 20 minutes at 2000×g prior to aliquoting. For serum-free medium, the presence of carrier proteins, e.g., 1% BSA, in the solution was used to prevent loss of analyte to the labware. Samples with extremely high levels of cytokines were diluted. Tissue culture supernatant samples were diluted at least 2-fold in diluent. Upon collection, samples were tested immediately or aliquots were frozen at ≦20° C. Samples were centrifuged at 2000 g for three minutes to remove particulates prior to sample preparation.

(iii) Dilution of Samples

For human serum, plasma, CSF, urine, and cell culture supernates, a minimum of 2-fold dilution in diluent was done.

(iv) Preparation of Controls

Controls were prepared in non-human animal matrix with spiked recombinant human analytes. The lyophilized controls were reconstituted in 250 uL of diluent and treated as a sample. Once reconstituted in 250 uL of diluent, the controls were stable for 30 days at 2-8° C.

(v) Preparation of Detection Antibody Solutions

Detection antibodies were obtained from Meso Scale Discovery (Rockville, Md.) as a 50× stock solution and the working detection antibody solution was 1×. Exposure of 1× detection antibody solution to light was avoided to prevent elevated assay background. Once prepared, the 1× detection antibody solution was kept in the dark.

For 1 plate of Panel 1, the following were combined:

-   -   1. 60 uL of 50× SULFO-TAG™ Anti-human IFN-gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human IL-1beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human IL-2 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human IL-4 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human IL-6 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IL-8 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human IL-10 antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-12p70 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human IL-13 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human TNFalpha antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 2, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-human GM-CSF antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human IL-1 alpha antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human IL-5 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human IL-7 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human IL-12/IL-23p40 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IL-15 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human IL-16 antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-17A antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human TNFbeta antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human VEGF-A antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 3, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-human Eotaxin antibody     -   2. 60 uL of 50× SULFO-TAG Anti-human MIP-1 beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-human MCP-4 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-human Eotaxin-3 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-human TARC antibody     -   6. 60 uL of 50× SULFO-TAG Anti-human IP-10 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-human MIP-1alpha antibody     -   8. 60 uL of 50× SULFO-TAG Anti-human IL-8 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-human MCP-1 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-human MDC antibody     -   11. 2400 uL Diluent 3 from Meso Scale Discovery (Rockville, Md.)         For 1 plate of Panel 4, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-rat IFN-gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-rat IL-2 antibody     -   3. 60 uL of 50× SULFO-TAG Anti-rat IL-4 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-rat IL-1 beta antibody     -   5. 60 uL of 50× SULFO-TAG Anti-rat IL-5 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-rat IP-6 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-rat KC/GRO antibody     -   8. 60 uL of 50× SULFO-TAG Anti-rat IL-10 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-rat IL-13 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-rat TNF alpha antibody     -   11. 2400 uL Diluent 40 from Meso Scale Discovery (Rockville,         Md.)         For 1 plate of Panel 5, the following were combined:     -   1. 60 uL of 50× SULFO-TAG Anti-mouse IFN gamma antibody     -   2. 60 uL of 50× SULFO-TAG Anti-mouse IL-1 beta antibody     -   3. 60 uL of 50× SULFO-TAG Anti-mouse IL-2 antibody     -   4. 60 uL of 50× SULFO-TAG Anti-mouse IL-4 antibody     -   5. 60 uL of 50× SULFO-TAG Anti-mouse IL-5 antibody     -   6. 60 uL of 50× SULFO-TAG Anti-mouse IP-6 antibody     -   7. 60 uL of 50× SULFO-TAG Anti-mouse KC/GRO antibody     -   8. 60 uL of 50× SULFO-TAG Anti-mouse IL-10 antibody     -   9. 60 uL of 50× SULFO-TAG Anti-mouse IL-12p70 antibody     -   10. 60 uL of 50× SULFO-TAG Anti-mouse TNF alpha antibody     -   11. 2400 uL Diluent 45 from Meso Scale Discovery (Rockville,         Md.)

(vi) Preparation of Read Buffer

Read Buffer T (also available from Meso Scale Discovery) is obtained as a 4× stock solution and the working solution was 2×. For 1 plate, equal parts (10 mL) of Read Buffer T (4×) was combined with deionized water (10 mL). A working solution of read buffer was prepared in advance and stored at room temperature in a tightly sealed container (stable for up to three years).

(vii) Preparation of MSD Plate

Multi-well plates (also available from Meso Scale Discovery) were pre-coated with capture antibodies (FIG. 1) and exposed to a proprietary stabilizing treatment to ensure the integrity and stability of the immobilized antibodies. Plates were used as delivered; no additional preparation (e.g., pre-wetting) was required.

Example 2. Assay Protocol

(i) Fifty (50) uL of diluted sample (standards, controls, or unknowns) per well were added. The plate was sealed with an adhesive plate seal and incubated for 2 hours with vigorous shaking (300-1000 rpm) at room temperature.

(ii) The plate was washed 3 times with 150-300 uL/well of PBS-T. Twenty-five (25) uL of detection antibody solution was added to each well. The plate was sealed with an adhesive plate seal and incubated for 2 hours with vigorous shaking (300-1000 rpm) at room temperature.

(iii) The plate was washed 3 times with 150-300 uL/well of PBS-T. One hundred fifty (150) uL of 2× Read Buffer T (Meso Scale Discovery, Rockville, Md.) was added to each well. The plate was analyzed in a SECTOR® Imager (Meso Scale Discovery, Rockville, Md.).

Example 3. Panel Verification

Assay development and evaluation of assay performance was executed utilizing industry and regulatory guidelines. During product development, kit components and protocols were developed and optimized to yield optimum product performance. The robustness of the assay protocol was evaluated to examine the boundaries of selected incubation times. Accelerated stability studies for calibrators, antibodies, and controls were performed during assay development and were augmented with real-time stability studies on complete kits out to 36 months from the date of manufacture. Verification of product design specifications was performed by evaluating standard curves, and a set of controls for each panel (also obtained from Meso Scale Discovery, Rockville, Md.) for three days by two independent analysts for a total of eight plates. Each plate was considered as a run. A summary of the standard curve data is shown in FIGS. 2(a)-(e) and Tables 4-8.

Intra- and inter-run precision and accuracy for a set of controls for each panel was evaluated for nine runs. Precision and accuracy were verified for each lot as part of the lot verification and quality control release. The typical specification for precision is a concentration CV of less than 20% for controls on both intra- and inter-day runs. As part of product verification, the performance of each panel was evaluated for spike and recovery and dilution linearity in serum, heparin plasma, EDTA plasma, citrate plasma, CSF, urine, and/or cell culture supernates. Native human analyte levels were measured in serum, heparin plasma, EDTA plasma, citrate plasma, CSF, and urine. Native rat and mouse analyte levels were measured in serum, heparin plasma, EDTA plasma, and urine.

Pooled human blood was stimulated in vitro with different stimuli (LPS and Zymosan and Peptidoglycan) and at the end of the stimulation period, plasma was isolated. In addition, for panels 1-3, THP-1 cell line was stimulated with LPS and at the end of stimulation, lysates were prepared. Freshly isolated PBMC were treated with different stimulating agents and supernates were isolated. The plasma, the cell lysates, and PBMC supernates were then evaluated for native human analyte levels using panels 1-3. For panel 4, rat macrophase cell line NR8383 was stimulated with LPS, PHA, and Pokeweed mitogen (PWM) and the cell lysate and cell culture supernates were isolated. The plasma, cell lysates, and cell culture supernates were evaluated for native rat analyte levels using panel 4. For panel 5, RAW cell line was stimulated with LPS and J774A.1 cell line was stimulated with LPS and PWM and at the end of stimulation, lysates were prepared. The plasma and cell lysates were then evaluated for native mouse analyte levels using panel 5.

FIG. 2(a)-(e) shows a standard curve graph that illustrates the dynamic range of each panel (panels 1-5, respectively).

TABLE 4 Panel 1 Typical Data IFNγ IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0      364 9.5 0   979 7.6  0.31    485 5.0 0.12   1435 5.2 1.2    856 5.1 0.49   2640 4.6 4.9   2120 2.7 2.0   7149 4.0 20     7559 1.8 7.8 25 643 3.5 78    29 285 1.1 31 97 601 2.3 313    114 776 2.0 125 385 790  4.3 1250    420 758 1.6 500 1 523 384   2.5 IL-2 IL-4 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     278 12.0 0   203 11.1  0.31   533 9.0 0.05   365 10.1 1.2   1257 5.4 0.21   933 8.1 4.9   3826 3.7 0.82   3008 6.6 20   13 922 3.2 3.3 12 155 2.3 78   55 350 3.2 13 47 521 2.0 313    207 898  2.8 53 178 863  2.5 1250    740 647  3.8 210 618 057  2.2 IL-6 IL-8 IL-10 Conc. Average % Conc. Average % Conc. Average (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal % CV 0   219 8.0 0   208 12.0 0   232 10.7 0.16   404 9.4 0.12   370 5.4 0.08   420 9.8 0.63   982 4.8 0.49   858 4.3 0.30   937 5.4 2.5   3137 3.6 2.0   2724 2.7 1.2   2970 3.1 10 12 068 2.7 7.8 10 113 2.5 4.8 10 699 4.3 41 49 674 3.4 31 41 493 2.2 19 43 376 3.5 163 215 238  3.7 125 169 766  2.6 78 163 839  2.1 650 930 463  2.7 500 722 931  2.2 310 568 597  2.3 L-12p70 IL-13 INFα Conc. Average % Conc. Average % Conc. Average (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal % CV 0   267 15.5 0   188 18.5 0   138 19.2 0.10   327 13.1 0.11   241 15.1 0.08   266 12.8 0.41   511 7.2 0.45   405 9.3 0.32   606 3.9 1.6   1348 6.1 1.8   1030 3.7 1.3   1960 3.7 6.6   4787 3.6 7.3   3543 2.0 5.2   7100 2.5 25 18 351 2.3 29 17 828 2.1 21 29 253 2.6 105 69 571 4.3 118 110 781  4.4 83 118 437  4.7 420 250 748  2.8 470 587 447  1.7 330 508 866  2.7

TABLE 5 Panel 2 Typical Data GM-CSF IL-1α Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     221 14.3 0   401 11.9  0.24   355 7.5 0.1   531 7.5  0.98   781 5.5 0.4   852 5.8 3.9   2374 4.8 1.4   2167 8.4 16     9635 4.3 5.8   7368 5.1 63   35 827 3.4 23 27 075 5.5 250    139 828  3.4 93 110 306  3.2 1000    472 016  3.2 370 394 888  3.6 IL-5 IL-7 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     571 10.5 0   235 12.6 0.2   833 8.7 0.2   342 9.5 0.8   1438 7.4 0.7   712 6.1 3.1   3961 5.8 2.9   2107 5.1 12   14 364 7.9 12   8770 6.3 49   52 918 3.4 47 32 322 3.2 198    198 664  3.8 188 137 340  2.8 790    639 511  5.7 750 581 986  2.1 IL-12/IL-23 p40 IL-15 IL-16 Conc. Average % Conc. Average % Conc. Average (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal % CV 0   285 8.7 0   227 8.3 0   264 11.3 0.7   431 7.1 0.2   338 8.6 0.61   326 11.4 2.9   874 3.5 0.7   681 6.1 2.4   477 6.6 12   2531 3.8 2.7   1954 4.6 9.8   1090 4.8 47 10 105 6.5 11   7840 7.0 39   4481 6.4 188 36 783 5.9 44 28 139 3.5 156 15 741 2.1 750 144 847  2.8 175 105 824  5.3 625 82 935 4.1 3000 512 130  5.5 700 464 580  3.0 2500 436 497  4.5 IL-17A TNFβ VEGF Conc. Average % Conc. Average % Conc. Average (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal % CV 0   144 28.9 0   248 10.9 0   476 5.2 1.2   290 10.3 0.1   456 6.1 0.3   547 8.9 4.8   821 8.1 0.6   1156 3.8 1.0   681 5.4 19   2719 4.3 2.4   3813 3.0 4.2   1235 3.5 76 12 286 6.8 10 15 143 2.4 17   4187 7.1 304 45 447 4.4 38 57 815 2.1 67 14 990 3.7 1218 190 122  2.5 153 233 155  1.3 268 93 227 3.4 4870 684 182  4.4 610 890 796  3.2 1070 517 033  3.1

TABLE 6 Panel 3 Typical Data Eotaxin MIP-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     189 13.2 0    91 39.9  0.37   215 7.6 0.24   160 16.9 1.5   252 14.5 1.0   313 5.9 5.9   430 6.3 3.9   1033 3.9 23     2408 5.8 16   5293 3.0 94   21 595 1.8 63 35 620 2.3 375    159 348  1.8 250 210 203  3.4 1500    813 344  3.4 1000 834 900  4.2 Eotaxin-3 TARC Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     114 35.7 0   283 16.7 1.2   208 10.7 0.37   546 8.5 4.9   501 5.3 1.5   1409 3.5 20     1610 3.4 5.9   4580 3.0 78     6029 1.6 23 18 048 4.4 313    23 154 3.0 94 69 091 2.7 1250    85 026 5.2 375 269 113  2.7 5000    283 990  4.9 1500 873 335  2.9 IP-10 MIP-1α IL-8 Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV 0    58 29.3 0   141 18.3 0   127 21.5 0.6   435 7.1 0.2   170 16.9 18   218 23.5 2.4   1532 4.7 1.0   185 14.5 71   338 10.1 10   5855 3.5 3.9   297 14.3 283   883 7.6 39 22 406 3.8 15   1023 5.1 1131   4028 4.8 156 84 024 4.2 62   7812 2.7 4525 31 569 7.4 625 264 057  3.7 248 69 584 4.2 18100 314 805  4.6 2500 437 795  7.1 990 454 451  3.0 72400 1 784 204   3.3 MCP-1 MDC MCP-4 Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV 0   171 17.8 0   152 10.3 0    71 29.8 0.1   277 11.1 2   288 6.0 0.2   121 28.7 0.5   654 6.6 10   677 3.3 0.6   120 32.8 2.0   2010 6.4 39   2273 4.1 2.4   187 16.9 7.8   8243 6.1 156   9996 3.0 10   976 8.6 31 33 293 4.1 625 59 008 4.3 39   9827 3.8 125 134 498  3.3 2500 406 977  4.2 156 80 691 3.8 500 547 716  8.3 10000 1 763 220   3.8 625 494 877  1.7

TABLE 7 Panel 4 Typical Data IFNγ IL-2 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     462 11    0   334 13 1.2   663 6.8    12   330 16 4.9   1383 6.3    49   425 8.8 20     4056 3.8   195   747 5.9 78   15 075 2.7   781   1969 6.1 313    65 765 3.8   3125   7571 5.7 1250    318 965  2.3 1 2500 32 383 4.3 5000    1 334 897   4.5 50 000 147 984  7.6 IL-4 IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     281 11 0   523 14 0.2   348 11 2.6   506 13 1.0   499 7.9 11   542 9.2 4.0   1184 6.3 42   683 7.6 16     3974 6.4 169   1508 2.9 64   21 355 4.2 675   4915 2.9 255    132 949  3.7 2700 20 813 2.8 1020    660 873  4.9 10800 93 359 4.4 IL-5 IL-6 KC/GRO Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV    0   218 14    0   491 7.4 0   648 9.9    2.4   220 15    3.0   485 8.8 0.7   687 5.6    9.8   250 9.0    12   542 7.2 2.7   714 6.0    39   400 9.0    48   729 9.0 11   781 5.4   156   1477 6.6   194   1565 5.8 43   1228 6.8   625 11 226 6.1   775   5186 5.7 174   5568 5.0   2500 75 782 4.3   3100 22 199 5.6 695 49 356 3.9 10 000 295 607  6.3 12 400 108 108  11 2780 387 251  6.6 IL-10 IL-13 TNFα Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV    0   596 14 0   275 16 0   208 17    4.9   583 20 0.4   269 17 0.3   259 9.9    20   858 16 1.6   336 9.1 1.1   404 10    78   1630 7.9 6.3   563 8.6 4.5   993 6.1   313   5131 6.7 25   1544 3.6 18   3323 4.8   1250 19 039 5.2 100   7033 4.5 73 15 943 2.8   5000 73 865 4.3 400 44 979 2.3 290 92 423 3.7 20 000 278 070  6.1 1600 299 022  6.2 1160 535 091  4.1

TABLE 8 Panel 5 Typical Data IFNγ IL-1β Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     311 14.5 0   284 12.4  0.20   821 5.6 0.34   562 6.3  0.78   2443 2.4 1.4   1462 3.6 3.1   8641 2.3 5.5   4909 2.6 13   35 510 1.9 22 1 9280 2.1 50   137 358  1.8 88 7 7541 2.2 200    504 897  2.2 350 307 686  1.9 800    1 437 412   2.2 1400 1 149 886   1.9 IL-2 IL-4 Conc. Average Conc. Average (pg/mL) Signal % CV (pg/mL) Signal % CV 0     289 12.0 0   495 8.9  0.51   513 8.2 0.34   732 7.3 2.1   1232 5.4 1.4   1529 4.0 8.2   4094 3.6 5.5   4351 2.9 33   15 136 3.5 22 17 081 1.6 131    64 545 3.4 88 65 254 2.7 525    264 160  2.3 350 245 316  2.6 2100    939 169  1.6 1400 798 882  2.1 IL-5 IL-6 KC/GRO Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV 0   145 21.6 0   369 7.8 0   307 10.3 0.2   412 10.6 1.1   544 6.0 0.39   460 9.1 0.78   1298 4.2 4.4   1107 5.9 1.6   1129 2.6 3.1   4826 2.6 18   3321 2.3 6.3   3695 1.9 13 19 027 3.0 70 12 162 1.8 25 13 975 2.8 50 79 736 3.9 281 50 520 2.8 100 65 482 2.6 200 336 576  2.9 1125 225 797  2.2 400 335 078  1.9 800 1 282 609   2.9 4500 1 085 547   2.8 1600 1 609 664   2.1 IL-10 IL-12p70 TNFα Conc. Average % Conc. Average % Conc. Average % (pg/mL) Signal CV (pg/mL) Signal CV (pg/mL) Signal CV 0   763 10.1 0   372 7.6 0   883 5.8 0.63   819 5.9 6.4   453 11.1 0.12   985 5.5 2.5   1125 6.2 26   684 5.1 0.49   1291 4.3 10   2349 3.1 103   1754 2.9 2   2592 3.1 41   7758 3.4 413   6261 2.6 7.8   7460 3.4 163 29 289 2.2 1650 30 330 2.9 31 29 633 2.9 650 121 581  3.7 6600 163 885  2.8 125 128 475  3.0 2600 504 589  4.3 26400 731 703  2.9 500 582 743  3.3

The lower limit of detection (LLOD) is a calculated concentration based on a signal 2.5 standard deviations above the background (zero calibrator blank). The LLOD shown in Tables 9-13 for each panel was calculated based on 8-9 runs.

TABLE 9 Panel 1 LLOD IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Median LLOD 0.22 0.05 0.12 0.03 0.07 0.01 0.03 0.13 0.27 0.06 (pg/mL) LLOD Range 0.10-0.37 0.02-0.08 0.07-0.13 0.02-0.04 0.06-1.0 0.01-0.01 0.02-0.05 0.09-0.19 0.21-0.43 0.05-0.10 (pg/mL)

TABLE 10 Panel 2 LLOD IL-12/IL- GM-CSF IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Median LLOD 0.14 0.08 0.10 0.15 0.40 0.14 1.60 0.77 0.06 0.9 (pg/mL) LLOD Range 0.10-0.34 0.05-0.29 0.08-0.28 0.11-0.22 0.30-0.58 0.08-0.19 0.98-2.77 0.50-2.70 0.04-0.12 0.75-1.39 (pg/mL)

TABLE 11 Panel 3 LLOD Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Median LLOD 3.2 0.34 1.2 0.13 0.13 1.4 5.8 0.09 2.6 2.3 (pg/mL) LLOD Range 2.2-4.0 0.42-0.89 0.79-3.1 0.00-0.22 0.09-0.22 1.4-2.3 31-62 0.07-0.20 1.7-2.8 1.6-2.7 (pg/mL)

TABLE 12 Panel 4 LLOD IFNγ IL-2 IL-4 IL-1β IL-5 IL-6 KC/GRO IL-10 IL-13 TNFα Median LLOD 0.7 57 0.7 35 27 23 21 14 3.7 0.9 (pg/mL) LLOD Range 0.4-2.7 38-126 0.4-1.0 16-80 19-37 16-41 19-30 8.9-20 2.7-8.6 0.5-1.5 (pg/mL)

TABLE 13 Panel 5 LLOD IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα Median 0.042 0.11 0.22 0.19 0.056 0.606 0.22 1.1 8.9 0.15 LLOD (pg/mL) LLOD 0.025-0.084 0.093-0.171 0.165-0.338 0.099-0.343 0.05-0.099 0.486-1.075 0.185-0.373 0.518-3.19 7.598-14.254 0.109-0.548 Range (pg/mL)

Controls were made by spiking calibrator into non-human animal matrix for panels 1-3, rat serum for panel 4, and mouse serum for panel 5, at levels throughout the range of the assay. Analyte levels were measured using a minimum of 3 replicates on 3 runs over 3 days. Average intra-run % CV is the average % CV of the control replicates within an individual run. Inter-run % CV is the variability of controls across a selected number of runs. Inter-lot % CV is the variability of controls across a selected number of kit lots.

TABLE 14 Panel 1 Average Average Inter- Conc. Intra-run run Inter-lot Control Runs (pg/mL) % CV % CV % CV IFNγ High 9 1941 1.9 6.8 4.8 Mid 9 203 1.9 8.3 Low 9 16 4.8 7.8 IL-1β High 9 107 2.6 5.3 Mid 9 11 1.8 6.5 Low 9 7 3.4 9.8 IL-2 High 9 986 2.2 3.2 Mid 9 99 2.2 4.8 Low 9 9 4.9 13 IL-4 High 9 294 1.7 6.2 Mid 9 32 4.4 6.7 Low 9 4 3.5 5.8 IL-6 High 9 801 3.4 5.8 Mid 9 76 3.0 4.6 Low 9 6 4.1 7.0 IL-8 High 9 613 2.3 5.2 Mid 9 60 1.8 6.1 Low 9 8 3.4 8.4 IL-10 High 9 372 2.2 2.8 Mid 9 39 1.5 5.5 Low 9 4 3.3 8.6 IL-12p70 High 9 467 4.4 6.4 Mid 9 51 3.2 6.6 Low 9 5 3.9 4.3 IL-13 High 9 657 3.0 8.6 Mid 9 74 2.7 13.8 Low 9 5 6.3 11.6 TNFα High 9 270 4.0 7.2 Mid 9 24 3.7 9.6 Low 9 3 3.7 12.0

TABLE 15 Panel 2 Average Average Inter- Conc. Intra-run run Inter-lot Control Runs (pg/mL) % CV % CV % CV GM-CSF High 9 506 4.0 22.1 N/A Mid 9 53 2.6 20.8 N/A Low 9 5 4.8 16.8 N/A IL-1α High 9 144 3.4 11.8 N/A Mid 9 15 3.2 12.4 N/A Low 9 2 5.7 13.8 N/A IL-5 High 9 450 4.2 16.6 N/A Mid 9 45 2.4 18.2 N/A Low 9 4 4.4 16.0 N/A IL-7 High 9 437 3.3 9.7 N/A Mid 9 44 2.9 4.8 N/A Low 9 5 3.8 4.6 N/A IL-12/IL-23 High 9 1631 3.7 10.9 N/A p40 Mid 9 173 2.4 9.9 N/A Low 9 17 3.9 8.0 N/A IL-15 High 9 317 3.5 26.0 N/A Mid 9 35 3.8 31.1 N/A Low 9 4 4.7 25.6 N/A IL-16 High 9 1965 2.3 19.8 N/A Mid 9 166 1.7 23.7 N/A Low 9 19 5.2 25.8 N/A IL-17A High 9 2662 5.3 20.0 N/A Mid 9 256 4.3 18.1 N/A Low 9 25 4.3 16.2 N/A TNFβ High 9 298 4.4 27.2 N/A Mid 9 30 3.0 27.7 N/A Low 9 3 3.0 25.0 N/A VEGF High 9 766 2.6 18.3 N/A Mid 9 64 2.7 12.1 N/A Low 9 8 5.9 5.4 N/A

TABLE 16 Panel 3 Average Average Inter- Conc. Intra-run run Inter-lot Control Runs (pg/mL) % CV % CV % CV Eotaxin High 9 2889 2.3 5.9 Mid 9 312 2.3 4.5 Low 9 34 12.8 3.4 MIP-1β High 9 2071 3.0 5.3 Mid 9 222 1.4 4.8 Low 9 20 4.4 5.7 Eotaxin-3 High 9 13931 2.1 6.5 Mid 9 1025 4.4 5.0 Low 9 131 7.0 9.5 TARC High 9 3240 4.6 6.2 Mid 9 332 3.0 3.6 Low 9 34 4.3 8.4 IP-10 High 9 5858 9.3 14.4 Mid 9 435 3.5 5.3 Low 9 51 4.2 10.2 MIP-1α High 9 2253 1.8 4.0 Mid 9 219 1.6 5.4 Low 9 26 10.4 8.8 IL-8 High 9 125226 2.3 10.7 Mid 9 44664 1.3 13.2 Low 9 4830 2.1 18.8 MCP-1 High 9 1066 5.1 8.4 Mid 9 113 5.0 4.4 Low 9 11 6.3 6.2 MDC High 9 25521 4.6 5.3 Mid 9 1548 4.1 5.2 Low 9 197 4.1 8.3 MCP-4 High 9 1349 3.0 5.3 Mid 9 170 2.5 5.1 Low 9 14 12.3 14.6

TABLE 17 Panel 5 Average Average Inter- Inter- Conc. Intra-run run lot Control Runs (pg/mL) % CV % CV % CV IFNγ High 9 305 2.1 4.5 4.8 Mid 9 722 2.2 9.6 Low 9 23 1.3 6.6 IL-1β High 9 826 2.0 3.4 Mid 9 928 2.0 7.5 Low 9 53 1.7 5.2 IL-2 High 9 2,092 2.3 3.8 Mid 9 2,293 2.2 7.8 Low 9 80 2.4 5.6 IL-4 High 9 759 3.8 6.0 Mid 9 836 2.9 9.3 Low 9 70 2.4 6.8 IL-5 High 9 849 2.0 4.2 Mid 9 981 2.8 7.0 Low 9 36 2.2 4.8 IL-6 High 9 115 2.4 3.7 Mid 9 400 3.5 11 Low 9 26 2.5 5.4 KC/GRO High 9 776 3.1 3.4 Mid 9 752 2.7 6.2 Low 9 106 3.4 4.8 IL-10 High 9 3,370 3.1 4.3 Mid 9 4,167 3.1 7.6 Low 9 627 2.4 6.1 IL-12p70 High 9 7,821 4.7 7.8 Mid 9 26,735 7.0 9.9 Low 9 3,193 4.5 12 TNFα High 9 448 2.5 5.0 Mid 9 479 2.1 7.0 Low 9 22 3.0 5.1

To assess linearity in panels 1-3, normal individual human serum, EDTA plasma, heparin plasma, citrate plasma, and CSF samples from a commercial source were spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold before testing. Normal individual human urine was spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, and 16-fold. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 2-fold dilution for panels 1-2 and a 4-fold dilution for panel 3 (see equation below).

To assess linearity in panel 4, normal rat serum. EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were spiked with recombinant calibrators and diluted 4-fold, 8-fold, 16-fold, and 32-fold before testing. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 4-fold dilution.

To assess linearity in panel 5, normal mouse serum, EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were spiked with recombinant calibrators and diluted 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, and 64-fold before testing. Percent recovery at each dilution was calculated by dividing the dilution adjusted calculated concentration by the expected concentration, i.e., the calculated dilution adjusted concentration at 2-fold dilution.

The average percent recovery shown below is based on samples within the quantitative range of the assay.

${\% \mspace{14mu} {Recovery}} = {\left( \frac{Measured}{Expected} \right)*100}$

TABLE 18 Panel 1 IFNγ IL-1β IL-2 IL-4 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 105 95-109 106 100-118  91 78-121 106 93-128 (N = 12) 8 101 91-112 103 92-129 91 71-158 103 87-133 16 100 92-119 102 85-121 94 63-196 107 94-139 32 98 87-120 105 85-136 107 63-283 103 88-135 64 102 88-125 110 88-143 120 63-402 108 88-142 EDTA 4 108 101-124  108 100-115  92 81-121 110 96-129 Plasma 8 107 93-131 106 94-119 91 75-157 108 86-140 (N = 12) 16 108 89-135 107 85-125 96 69-210 111 80-153 32 103 79-135 107 81-128 105 66-282 105 71-140 64 109 80-141 112 84-136 116 65-412 109 76-152 Heparin 4 106 97-116 109 100-123  94 76-122 107 92-128 Plasma 8 101 90-110 108 99-118 96 70-161 104 84-142 (N = 12) 16 102 89-112 106 93-122 102 65-206 108 82-151 32 98 84-112 108 98-124 110 61-277 105 81-149 64 101 83-124 109 93-137 125 54-435 110 81-157 Citrate 4 102 95-107 100 92-105 79 61-115 103 91-109 Plasma 8 97 87-104 99 94-107 74 50-146 99 89-110 (N = 10) 16 94 85-105 96 89-109 71 46-174 98 86-115 32 89 80-104 94 80-113 72 46-191 95 82-124 64 91 81-106 94 84-113 73 45-207 99 85-129 Urine 4 95 92-100 98 93-102 87 75-103 97 93-102 (N = 5) 8 88 79-91  91 85-96  79 63-96  98 90-110 16 87 83-90  90 88-94  78 58-100 99 90-112 Cell Culture 4 102 95-105 100 95-105 87 85-88  101 96-108 Supernates 8 97 92-103 98 90-104 83 78-88  100 94-107 (N = 6) 16 96 89-105 89 83-97  77 73-81  102 94-114 32 88 82-94  86 81-99  75 71-77  95 87-103 64 90 80-97  85 75-94  70 65-77  94 78-104 IL-6 IL-8 IL-10 IL-12p70 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 105 95-113 97 89-103 102 93-108 104 99-117 (N = 12) 8 106 96-124 93 86-104 102 91-114 102 90-111 16 104 89-117 88 78-100 97 89-113 104 91-119 32 104 93-118 92 79-106 102 90-123 105 93-118 64 110 95-127 95 79-110 104 89-124 110 94-131 EDTA 4 104 99-115 97 94-104 106 100-116  106 94-126 Plasma 8 106 97-121 92 86-99  106 93-120 107 95-133 (N = 12) 16 106 90-132 90 74-104 103 82-119 108 92-142 32 105 88-133 90 71-102 107 81-131 108 87-152 64 113 93-144 95 72-108 106 81-132 114 88-154 Heparin 4 108 100-130  99 92-104 101 91-108 105 96-115 Plasma 8 105 94-121 94 83-101 101 86-111 102 94-116 (N = 12) 16 106 84-121 92 83-100 97 81-111 106 93-131 32 104 81-121 94 80-102 100 83-112 103 93-126 64 108 89-130 96 82-108 99 81-118 109 92-155 Citrate 4 107 95-169 97 93-107 97 94-100 101 90-115 Plasma 8 112 83-264 89 83-97  94 87-108 96 84-111 (N = 10) 16 127 85-416 85 74-95  88 74-105 94 78-109 32 136 78-550 85 72-97  90 75-114 91 73-111 64 156 79-702 86 73-99  88 71-111 95 74-118 Urine 4 106 104-104  98 94-104 95 91-99  94 91-100 (N = 5) 8 102 100-100  93 82-101 90 86-93  90 84-98  16 100 101-101  92 84-98  86 83-87  94 81-106 Cell Culture 4 97 80-107 96 90-98  101 99-104 88 77-95  Supernates 8 93 84-101 92 89-98  100 95-106 86 81-89  (N = 6) 16 87 79-95  85 80-92  94 87-100 84 81-89  32 84 74-92  86 82-95  94 87-101 79 70-85  64 84 75-96  84 78-91  90 83-98  81 74-88  IL-13 TNFα Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4 88 79-103 98 88-107 (N = 12) 8 79 70-101 95 89-111 16 73 63-101 90 82-110 32 74 62-108 94 85-115 64 79 65-114 95 86-119 EDTA 4 90 84-102 97 93-103 Plasma 8 83 74-111 94 87-103 (N = 12) 16 77 62-117 89 77-103 32 76 53-117 92 77-104 64 81 59-127 94 76-107 Heparin 4 93 83-109 99 89-105 Plasma 8 84 72-114 96 81-101 (N = 12) 16 79 63-111 93 78-102 32 77 60-114 94 78-97  64 82 62-126 95 77-105 Citrate 4 87 81-95  95 90-101 Plasma 8 78 67-88  89 80-106 (N = 10) 16 66 57-78  83 70-108 32 65 57-79  84 71-107 64 67 60-88  84 71-108 Urine 4 88 82-95  87 82-92  (N = 5) 8 77 68-84  80 76-84  16 75 62-81  76 68-83  Cell Culture 4 89 84-98  86 80-91  Supernates 8 79 74-83  79 74-85  (N = 6) 16 70 65-73  72 63-81  32 67 64-71  73 67-80  64 68 65-73  71 61-78 

TABLE 19 Panel 2 CM-CSF IL-1α IL-5 IL-7 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 108 91-136 118 98-170 108 89-169 96  84-120 (N = 11) 8 98 77-141 120 65-220 101 76-134 85  65-104 16 94 68-145 138 59-320 94 74-129 82  64-107 32 89 64-144 175 63-621 99 73-129 78  66-108 64 92 66-143 209 75-834 99 76-125 82  71-122 EDTA 4 100 89-122 103 85-137 102 93-116 91 86-95 Plasma 8 91 80-123 101 78-182 98 82-124 82 75-91 (N = 11) 16 87 74-119 100 66-202 90 69-114 78 70-88 32 80 66-103 104 62-247 88 66-115 72 62-86 64 81 68-106 114 65-276 86 65-107 76 62-84 Heparin 4 102 88-135 105 88-139 106 87-123 98  84-106 Plasma 8 93 78-142 102 74-181 103 86-127 91 73-99 (N = 11) 16 93 72-152 109 63-258 96 77-127 89 72-99 32 89 72-144 114 59-294 97 73-124 87  65-100 64 93 74-159 134 66-422 96 65-122 93  66-110 Citrate 4 97 95-99  120 92-156 98 89-114 92  88-100 Plasma 8 85 81-88  129 88-209 92 78-115 82 73-94 (N = 10) 16 81 72-86  139 86-253 86 73-112 79 70-93 32 74 65-80  140 84-266 82 71-109 74 66-86 64 75 69-83  145 83-320 78 68-111 75 68-82 Urine 4 114 104-122  131 73-167 108 96-132 107  99-112 (N = 5) 8 122 104-127  116 57-157 121 111-134  111 101-116 16 131 127-135  102 91-127 132 124-155  124 114-132 Cell Culture 4 93 86-98  110 95-124 94 89-98  88 85-91 Supernates 8 91 86-98  109 96-137 89 87-92  89 83-93 (N = 6) 16 89 83-101 101 89-116 83 80-85  86 82-91 32 88 83-95  105 96-122 83 81-85  89 82-97 64 91 84-99  104 88-120 80 78-83  92  86-100 IL-12/IL-23 p40 IL-15 IL-16 IL-17A Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 101 90-128 90  79-115 95 86-103 104 74-128 (N = 11) 8 91 65-114 85 69-94 88 72-99  95 58-108 16 90 66-114 80 62-94 86 73-101 90 64-95  32 87 71-107 83 73-98 91 77-101 87 72-96  64 89 78-119 83  70-101 98 83-122 88 77-100 EDTA 4 98 85-110 85 77-93 93 81-104 101 92-111 Plasma 8 91 72-106 82 75-94 79 68-93  96 86-108 (N = 11) 16 87 68-102 77 69-82 74 58-88  93 79-111 32 81 62-97  74 62-83 77 60-100 86 68-106 64 83 61-99  73 63-81 80 60-106 87 67-102 Heparin 4 107 91-133 83 72-93 97 79-115 102 83-110 Plasma 8 101 82-131 78 64-87 89 64-102 95 84-108 (N = 11) 16 103 80-145 75 64-84 87 57-107 94 82-104 32 99 73-130 74 58-89 93 58-120 87 76-97  64 103 78-144 75 61-86 99 63-123 89 75-109 Citrate 4 102 95-112 85 76-93 92 86-99  102 93-128 Plasma 8 96 77-113 79 70-90 81 76-91  94 81-143 (N = 10) 16 97 82-116 77 68-88 75 67-85  93 75-158 32 90 72-113 75 67-87 76 67-91  89 70-156 64 93 74-110 72 64-81 78 70-93  90 73-152 Urine 4 116 80-142 107  77-136 134 118-163  113 95-148 (N = 5) 8 127 95-155 120  98-143 149 127-183  124 99-170 16 146 116-181  131 107-156 163 142-199  136 109-187  Cell Culture 4 103 94-109 80 78-81 101 95-110 89 83-91  Supernates 8 95 90-101 80 76-85 99 95-103 81 74-89  (N = 6) 16 93 86-100 82 77-87 96 91-114 79 75-84  32 90 87-94  84 77-90 107 97-129 79 73-84  64 94 90-100 90 86-95 112 102-140  84 76-89  TNFβ VEGF Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4 106 86-143 106 91-121 (N = 11) 8 100 76-116 95 77-113 16 98 72-115 96 70-122 32 99 69-120 118 74-170 64 97 71-116 145 82-213 EDTA 4 100 93-108 92 74-107 Plasma 8 94 83-108 83 69-96  (N = 11) 16 85 74-104 77 65-90  32 80 68-92  84 68-101 64 79 66-95  95 71-121 Heparin 4 102 90-114 107 85-115 Plasma 8 99 83-115 94 80-110 (N = 11) 16 96 82-114 82 65-99  32 94 77-111 96 57-130 64 94 77-114 139 54-234 Citrate 4 117 106-143  96 87-108 Plasma 8 122 102-159  89 77-102 (N = 10) 16 120 94-167 89 69-114 32 113 82-169 102 71-137 64 108 81-153 116 76-148 Urine 4 111 94-119 106 103-111  (N = 5) 8 120 97-130 108 105-115  16 143 105-176  112 105-119  Cell Culture 4 87 86-89  89 87-90  Supernates 8 84 82-86  79 76-82  (N = 6) 16 81 79-83  75 70-80  32 81 79-83  76 72-82  64 82 77-85  77 72-82 

TABLE 20 Panel 3 Eotaxin MIP-1β Eotaxin-3 TARC Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 2 88 73-106 97 55-117 113 88-134 92 80-108 (N = 12) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 104 100-112  105 91-150 93 86-106 94 89-102 16 105 95-124 113 87-192 90 74-105 94 83-108 32 110 85-145 118 87-226 96 73-119 92 83-111 64 111 81-146 120 81-245 100 73-128 98 81-126 EDTA 2 91 84-102 93 59-107 131 95-163 94 79-109 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 104 86-115 108 99-143 78 61-96  95 85-106 16 106 92-119 114 96-177 72 59-93  95 78-112 32 105 87-135 123 96-207 73 60-103 88 72-127 64 105 85-151 122 91-220 77 64-115 92 76-131 Heparin 2 89 75-119 94 61-109 112 97-143 75 61-103 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 108 101-119  107 96-138 89 78-100 110 95-121 16 120 80-135 111 93-173 89 71-104 118 67-135 32 135 86-157 117 95-197 92 60-125 120 80-139 64 145 87-170 113 90-202 101 66-138 126 80-170 Citrate 2 95 85-104 106 99-117 122 111-137  97 89-141 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 10) 8 102 98-105 99 91-105 86 74-98  85 60-102 16 102 95-110 95 89-101 78 66-87  78 48-97  32 99 88-110 95 86-106 79 64-90  72 44-86  64 98 83-118 93 82-110 87 71-100 74 43-89  Urine 2 96 78-116 88 63-107 106 97-119 92 78-110 (N = 5) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 106 102-110  105 101-109  97 92-102 94 87-108 16 114 108-116  109 103-114  94 87-99  92 85-110 Cell Culture 2 134 117-141  110 98-116 93 88-97  93 84-101 Supernates 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 6) 8 95 91-100 95 91-100 100 96-102 91 87-98  16 95 88-99  94 90-99  108 101-114  87 80-94  32 95 89-101 91 87-99  112 105-118  82 75-89  64 99 88-107 95 89-102 126 116-133  90 76-99  IP-10 MIP-1α IL-8 MCP-1 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 2 118 107-130 105 91-115 91 82-104 92 81-97  (N = 12) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 89 80-95 97 89-104 102 85-108 98 92-107 16 84 76-93 93 81-106 98 77-112 94 86-100 32 81 71-90 93 78-118 112 88-135 92 82-102 64 84 72-95 92 71-119 128 97-162 98 82-116 EDTA 2 117 104-149 103 73-112 93 84-108 99 93-107 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 89 81-96 99 90-111 90 79-100 92 83-97  16 86 73-99 97 88-113 87 75-100 88 78-98  32 86  71-100 98 86-116 98 83-109 88 77-98  64 91  75-106 97 80-119 112 94-127 95 83-108 Heparin 2 112  98-124 107 101-116  97 87-123 96 82-106 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 12) 8 89 80-98 95 90-100 91 76-113 96 87-105 16 86 75-97 92 80-103 82 66-99  92 81-106 32 84  69-101 90 76-107 88 65-116 90 79-105 64 95  75-110 88 71-109 100 75-140 94 76-112 Citrate 2 131  98-169 109 102-115  98 90-109 99 90-105 Plasma 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 10) 8 88 72-97 94 88-105 88 79-100 91 83-101 16 82 68-95 87 78-108 79 73-90  87 82-95  32 79 62-89 83 74-111 87 79-102 83 74-89  64 84  65-101 79 70-108 97 87-114 86 78-94  Urine 2 93  75-102 111 104-115  117 107-126  95 91-101 (N = 5) 4 100 N/A 100 N/A 100 N/A 100 N/A 8 97  93-106 96 95-99  87 78-94  98 95-102 16 97  87-109 92 89-97  77 68-85  96 91-103 Cell Culture 2 179 127-256 120 104-128  89 84-94  98 93-108 Supernates 4 100 N/A 100 N/A 100 N/A 100 N/A (N = 6) 8 73 55-84 90 85-94  96 89-113 95 89-98  16 68 60-88 85 78-94  89 83-95  94 89-101 32 63 52-77 80 72-89  93 82-97  88 81-91  64 68 57-81 81 73-95  107 95-121 98 90-103 MDC MCP-4 Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 2 109 101-121 84 78-93 (N = 12) 4 100 N/A 100 N/A 8 89 81-96 113 98-127 16 79 66-87 116 97-141 32 72 57-83 125 99-168 64 69 48-81 123 97-174 EDTA 2 106  91-118 76 65-86 Plasma 4 100 N/A 100 N/A (N = 12) 8 93  83-101 115 102-144 16 84 69-94 122  97-163 32 79 65-89 126  89-188 64 76 64-87 125  83-193 Heparin 2 106  98-118 84 75-92 Plasma 4 100 N/A 100 N/A (N = 12) 8 90 78-99 112  99-125 16 78 62-89 118  94-154 32 73 57-83 124  87-181 64 70 57-82 127  86-209 Citrate 2 105  77-130 89 81-98 Plasma 4 100 N/A 100 N/A (N = 10) 8 88 69-94 102  95-108 16 74 57-85 96  90-106 32 68 55-75 94  86-103 64 64 52-69 95  86-110 Urine 2 92  71-112 96 95-97 (N = 5) 4 100 N/A 100 N/A 8 86  61-102 105 103-107 16 75 54-89 108 106-113 Cell Culture 2 191 158-211 91 84-96 Supernates 4 100 N/A 100 N/A (N = 6) 8 75 73-76 110 105-113 16 63 61-65 113 105-120 32 56 54-59 111 103-118 64 55 52-57 113 105-124

TABLE 21 Panel 4 IFNγ IL-2 IL-4 IL-1β Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 8 109% 100%-110% 100% 88%-107%  96%  91%-109% 106% 101%-119% (N = 5) 16 109%  87%-112% 103% 97%-109%  96%  88%-110% 118% 109%-126% 32 118% 106%-128% 102% 99%-106% 111% 106%-117% 125%  98%-163% EDTA 8 114% 100%-119% 102% 88%-105%  96%  92%-101% 112% 104%-120% Plasma 16 125% 107%-140% 110% 97%-124% 101%  97%-107% 135% 120%-1485  (N = 5) 32 127% 115%-136% 104% 96%-116% 105% 100%-112% 162% 102%-193% Heparin 8 116% 113%-119% 102% 100%-104%  112% 104%-119% 108%  97%-114% Plasma 16 130% 121%-147% 111% 101%-118%  118% 103%-124% 129% 115%-149% (N = 5) 32 141% 129%-162% 111% 104%-119%  131% 119%-139% 134% 111%-150% Urine 8 114% 100%-121% 102% 104%-105%   96%  92%-101% 112% 104%-120% (N = 5) 16 125% 107%-140% 110% 97%-124% 101%  97%-107% 135% 120%-148% 32 127% 115%-136% 104% 96%-116% 105% 100%-112% 162% 102%-193% Cell Culture 8 Supernates 16 (N = 4) 32 IL-5 IL-6 KC/GRO IL-10 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 8 100% 99%-106% 100% 95%-107%  98% 95%-103% 100% 99%-106% (N = 5) 16 101% 87%-113% 102% 87%-111%  97% 87%-102% 101% 87%-113% 32 106% 96%-113% 117% 96%-135% 105% 93%-113% 106% 96%-113% EDTA 8  96% 83%-108% 107% 98%-125%  96% 93%-100%  96% 83%-108% Plasma 16 101% 92%-124% 119% 88%-149% 101% 94%-108% 101% 92%-124% (N = 5) 32  85% 62%-97%  N/A N/A  93% 65%-106%  85% 62%-97%  Heparin 8 106% 101%-116%  136% 118%-154%  116% 104%-128%  106% 101%-116%  Plasma 16 110% 100%-127%  N/A N/A 127% 116%-148%  110% 100%-127%  (N = 5) 32 118% 107%-144%  N/A N/A 131% 119%-155%  118% 107%-144%  Urine 8  96% 83%-108% 107% 98%-125%  96% 93%-100%  96% 83%-108% (N = 5) 16 101% 92%-124% 119% 88%-149% 101% 94%-108% 101% 92%-124% 32  85% 62%-97%  N/A N/A  93% 65%-106%  85% 62%-97%  Cell Culture 8 Supernates 16 (N = 4) 32 IL-13 TNFα Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 8 132% 126%-141% 103%  96%-112% (N = 5) 16 151% 140%-165% 108% 100%-115% 32 N/A N/A 121% 108%-132% EDTA 8 118% 111%-125% 104% 100%-107% Plasma 16 146% 144%-156% 113% 107%-121% (N = 5) 32 N/A N/A 117% 102%-126% Heparin 8 124% 117%-131% 114% 108%-119% Plasma 16 154% 136%-197% 123% 116%-129% (N = 5) 32 163% 150%-200% 139% 135%-146% Citrate 8 118% 111%-125% 104% 100%-107% Plasma 16 146% 131%-156% 113% 104%-121% (N = 5) 32 N/A N/A 117% 102%-126% Urine 8 118% 111%-125% 104% 100%-107% (N = 6) 16 146% 131%-156% 113% 104%-121% 32 N/A N/A 117% 102%-126% Cell Culture 8 Supernates 16 (N = 4) 32

TABLE 22 Panel 5 IFNγ IL-1β IL-2 IL-4 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 103 94-115 108 99-122 106 99-115 144 128-154 (N = 0) 8 99 86-107 96 82-102 103 89-110 156 136-169 16 98 86-108 98 89-105 94 82-110 170 145-182 32 95 92-101 99 87-109 96 79-110 171 139-191 64 98 92-109 100 89-109 97 83-111 194 144-204 EDTA 4 101 90-112 103 98-109 95 92-106 123 114-129 Plasma 8 100 80-115 101 86-111 94 84-104 134 127-149 (N = 0) 16 106 78-155 100 85-112 90 78-105 147 138-157 32 103 79-124 100 83-115 93 82-108 149 133-177 64 114 80-157 104 79-125 94 79-109 160 143-198 Heparin 4 105 82-122 106 100-112  101 94-112 134 124-139 Plasma 8 95 81-123 102 97-109 95 90-104 147 138-152 (N = 3) 16 107 91-171 100 93-111 92 83-107 162 153-179 32 103 86-181 100 92-111 95 81-109 159 129-174 64 106 87-147 101 94-115 93 78-108 174 154-194 Citrate 4 99 83-109 103 95-109 102 94-111 120 105-131 Plasma 8 91 79-100 95 85-102 94 89-103 125 112-133 (N = 3) 16 88 71-103 92 83-101 90 85-99  133 112-149 32 83 71-96  89 75-104 97 74-97  129 105-143 64 96 74-101 91 73-108 97 97-115 134 111-155 Urine 4 99 93-104 104 93-110 105 97-115 122 110-128 (N = 6) 8 96 97-104 102 93-111 103 95-119 136 129-143 16 96 89-101 99 81-103 101 94-118 149 145-153 32 94 89-100 101 94-112 103 95-124 151 146-157 64 97 86-103 102 86-112 103 91-122 166 160-169 Cell Culture 4 103 100-105  105 101-108  96 93-100 112 108-118 Supernates 8 98 96-99  102 99-107 92 89-95  112 108-115 (N = 4) 16 100 96-104 102 99-108 88 87-90  115 112-122 32 95 94-97  101 97-106 92 91-93  112 109-115 64 100 96-105 104 101-107  93 87-96  119 111-125 IL-5 IL-6 KC/GRO IL-10 Sample Fold Average % % Recovery Average % % Recovery Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Recovery Range Recovery Range Serum 4 106 92-129 98 87-109 111 105-154 120 105-142  (N = 0) 8 101 95-124 90 72-102 110  96-146 117 102-135  16 99 94-126 90 74-100 106  95-136 119 105-136  32 94 93-116 94 73-112 112  98-145 117 101-143  64 100 96-124 98 74-113 110 103-143 126 107-159  EDTA 4 100 90-107 97 83-95  91 83-95 103 88-127 Plasma 8 98 79-110 91 72-93  85 72-93 101 103-127  (N = 0) 16 95 80-114 88 64-90  82 84-90 102 94-122 32 99 79-120 91 83-106 87  68-106 99 95-124 64 105 82-133 95 72-112 90  72-112 102 90-129 Heparin 4 100 89-113 95 76-111 93  76-111 111 92-131 Plasma 8 95 87-105 98 88-115 90  88-115 107 94-122 (N = 3) 16 93 85-110 97 85-118 88  85-118 109 95-124 32 93 78-121 101 85-122 88  85-122 105 93-129 64 95 81-131 104 84-125 88  84-125 111 92-131 Citrate 4 97 94-106 91 86-99  90 77-99 99 86-106 Plasma 8 97 76-93  83 72-97  79 64-94 89 75-99  (N = 3) 16 83 70-102 79 67-94  73 59-86 87 75-101 32 83 66-102 90 65-99  71 53-91 93 71-90  64 83 50-107 92 60-102 74 54-91 90 76-103 Urine 4 98 94-114 99 95-104 85 78-95 103 87-114 (N = 6) 8 97 99-115 94 83-106 70 59-79 98 84-109 16 91 75-106 93 81-101 64 53-75 94 74-111 32 93 92-106 96 85-105 63 49-77 91 82-106 64 95 91-105 101 92-108 64 50-78 95 85-108 Cell Culture 4 96 94-98  93 91-94  76 72-80 93 87-100 Supernates 8 93 92-97  90 88-92  69 63-72 84 79-92  (N = 4) 16 92 86-96  88 89-31  65 60-69 84 77-94  32 92 85-99  93 88-97  67 62-72 81 74-90  64 95 89-100 96 90-102 70 66-76 85 77-101 IL-12p70 TNFα Sample Fold Average % % Recovery Average % % Recovery Type Dilution Recovery Range Recovery Range Serum 4 98  84-112 111 110-124  (N = 1) 8 87  73-100 108 99-120 16 91 65-94 109 97-123 32 79 62-90 117 105-142  64 81 61-93 123 107-156  EDTA 4 93  87-101 90 90-110 Plasma 8 85 78-94 100 88-111 (N = 0) 16 81 75-89 96 84-112 32 81 68-92 102 85-124 64 86  69-102 105 89-138 Heparin 4 87 77-95 102 95-111 Plasma 8 77 66-89 100 89-109 (N = 1) 16 75 68-84 100 88-109 32 74 62-87 105 89-118 64 78 63-90 107 91-119 Citrate 4 96  86-105 99 92-106 Plasma 8 84 68-96 94 89-97  (N = 1) 16 79 52-94 91 85-95  32 76 46-98 92 89-97  64 79  44-103 93 87-98  Urine 4 108  96-122 90 91-107 (N = 6) 8 107  87-133 96 84-112 16 106  90-132 97 85-116 32 106  91-140 102 89-122 64 113 101-148 107 94-130 Cell Culture 4 94 92-98 93 92-94  Supernates 8 87 85-88 89 87-93  (N = 4) 16 83 79-88 89 84-93  32 81 78-87 93 88-96  64 85 79-93 96 92-100

Spike and recovery measurements of different sample types throughout the quantifiable range of the assays were evaluated. Multiple individual human serum, EDTA plasma, heparin plasma, citrate plasma, urine, and/or CSF samples from a commercial source and cell culture supernates were spiked with calibrators at three levels (high, mid, and low) and subsequently diluted two-fold.

TABLE 23 Panel 1 IFNγ IL-1β Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 17-20 100 91-126 6-7 88 81-99  (N = 12) 173-193 99 84-121 59-67 89 77-103 1835-1874 107 95-117 630-635 100 81-112 EDTA 17-17 101 84-114 6-6 96 64-119 Plasma 173-178 103 82-118 59-61 97 71-119 (N = 12) 1874-1902 103 78-127 635-665 97 71-125 Heparin 16-17 101 89-123 6-6 97 78-118 Plasma 171-178 102 85-121 61-62 94 72-114 (N = 12) 1871-1902 98 86-119 665-681 92 74-112 Citrate 16-20 103 94-113 6-7 97 71-117 Plasma 171-214 105 95-121 62-74 96 66-114 (N = 10) 1871-2108 97 91-109 681-735 90 72-104 Urine 19-20 101 94-108 7-7 96 88-107 (N = 5) 204-214 103 93-111 69-74 99 90-108 2076-2108 99 91-106 733-735 95 83-101 Cell Culture 11-13 107 86-122 4-5 112 87-125 Supernates 168-173 105 97-123 60-61 115 101-133  (N = 6) 1919-2037 93 88-102 718-762 96 92-104 IL-2 IL-4 Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 13-15 91 27-151 3-3 89 81-114 (N = 12) 131-147 93 23-151 26-29 93 78-106 1524-1556 107 33-155 253-261 103 93-115 EDTA 13-13 98 23-170 3-3 97 81-106 Plasma 131-141 97 14-169 26-27 98 81-117 (N = 12) 1556-1674 104 18-181 261-269 102 81-128 Heparin 12-13 106 22-149 2-3 96 87-116 Plasma 135-141 105 16-143 26-27 93 78-115 (N = 12) 1633-1674 110 21-148 269-278 92 74-110 Citrate 12-15 104 39-155 2-3 96 74-121 Plasma 135-161 105 34-158 26-32 96 70-119 (N = 10) 1633-1775 105 43-144 278-296 89 73-104 Urine 14-15 123 92-163 3-3 99 91-110 (N = 5) 153-161 122 95-156 30-32 100 90-112 1726-1775 115 88-145 295-296 97 87-110 Cell Culture  9-10 140 106-165  2-2 93 70-111 Supernates 133-148 134 111-160  25-28 97 82-115 (N = 6) 1637-1828 119 110-131  273-304 85 78-96  IL-6 IL-8 Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 7-8 80 59-92  1-1 94 87-104 (N = 12) 63-73 83 63-96  7-7 97 90-107 725-768 97 83-119 74-76 109 94-118 EDTA 6-7 89 72-107 1-1 101 85-114 Plasma 63-66 91 72-109 7-7 104 83-114 (N = 12) 748-768 96 77-122 76-81 108 81-122 Heparin 6-7 90 49-107 1-1 98 87-115 Plasma 66-72 84 39-112 7-7 106 89-124 (N = 12) 748-881 89 47-111 78-81 103 89-119 Citrate 7-8 89  8-111 1-1 100 85-109 Plasma 72-79 92 11-120 7-8 108 93-119 (N = 10) 880-881 83  8-107 78-86 105 92-115 Urine 8-8 98 88-106 1-1 104 97-114 (N = 5) 79-81 96 82-104 8-8 102 93-110 880-908 91 74-99  84-86 100 84-107 Cell Culture 4-5 114 86-133 0-1 113 84-130 Supernates 86-67 114 98-139 7-7 116 103-138  (N = 6) 853-904 97 91-103 88-88 104 96-112 IL-10 IL-12p70 Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 4-4 101 83-129 5-5 96 78-137 (N = 12) 39-44 101 91-123 47-53 66 73-158 408-433 113 100-123  495-499 100 85-113 EDTA 4-4 103 82-119 4-5 99 73-122 Plasma 39-41 102 81-117 47-48 97 75-118 (N = 12) 433-434 104 80-127 499-506 100 82-121 Heparin 4-4 105 90-124 4-4 103 78-126 Plasma 41-42 106 91-123 48-48 98 74-121 (N = 12) 434-466 103 91-122 506-519 99 70-122 Citrate 4-5 107 94-126 4-5 104 89-132 Plasma 42-50 106 94-126 48-59 100 83-127 (N = 10) 466-480 100 93-109 519-550 95 71-122 Urine 4-5 98 90-108 5-6 107 97-121 (N = 5) 48-50 98 87-108 57-59 107 92-117 480-480 94 79-104 550-556 104 89-121 Cell Culture 2-3 103 75-119 3-3 120 94-137 Supernates 38-42 104 90-123 48-49 111 93-135 (N = 6) 422-487 93 86-99  522-547 111 103-119  IL-13 TNFα Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 5-6 106 74-126 4-5 104 83-146 (N = 12) 45-50 118 77-205 40-45 107 84-166 552-564 125 89-139 457-460 110 79-125 EDTA 5-5 115 92-130 4-4 108 95-120 Plasma 45-46 120 88-142 40-42 112 96-125 (N = 12) 564-574 124 87-157 460-469 110 88-132 Heparin 5-5 111 81-134 4-4 111 99-131 Plasma 46-46 116 87-138 42-44 111 102-130  (N = 12) 574-596 114 81-128 469-509 108 96-122 Citrate 5-6 120 103-134  4-5 112 101-122  Plasma 46-55 127 111-149  44-51 114 102-131  (N = 10) 596-629 118 105-137  509-530 105 95-122 Urine 6-6 114 106-127  5-5 99 83-112 (N = 5) 55-55 124 117-132  51-51 103 84-119 629-633 119 98-127 530-539 110 91-120 Cell Culture 4-4 124 95-143 4-4 126 99-147 Supernates 46-54 135 116-169  47-54 127 114-154  (N = 6) 617-696 118 104-126  581-672 117 104-127 

TABLE 24 Panel 2 GM-CSF IL-1α Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 6-6 92 70-102 6-6 74 44-102 (N = 11) 60-61 91 66-104 60-69 70 22-107 563-580 90 65-108 623-626 71 18-90  EDTA 6-6 97 62-115 6-6 84 48-100 Plasma 60-60 96 62-120 60-63 88 24-112 (N = 11) 562-563 95 60-128 611-623 86 17-108 Heparin 6-6 91 51-120 6-6 69 11-105 Plasma  58-60- 91 64-127 59-63 69 14-110 (N = 11) 506-562 94 64-123 556-611 69 12-108 Citrate 6-6 109 97-118 6-7 65 27-94  Plasma 58-59 104 96-110 59-64 63 23-100 (N = 10) 506-507 112 98-132 550-556 64 22-101 Urine 6-6 120 112-129  7-7 94 91-98  (N = 5) 57-59 120 106-138  61-64 102 90-115 507-515 126 113-132  543-550 104 90-113 Cell Culture 11 101 93-114 4 97 91-105 Supernates 105 95 87-103 34 90 76-96  (N = 6) 1121 95 93-110 343 89 77-96  IL-5 IL-7 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 3-3 87 74-110 1-1 101 68-111 (N = 11) 31-32 87 63-107 12-13 100 74-112 300-332 85 54-105 124-131 100 83-117 EDTA 3-3 85 56-106 1-1 102 92-115 Plasma 31-35 85 57-101 12-12 106 92-119 (N = 11) 326-332 88 52-120 124-127 105 91-130 Heparin 3-3 84 57-107 1-1 87 76-113 Plasma 34-35 82 55-104 12-13 83 72-126 (N = 11) 326-361 85 61-112 126-127 88 75-124 Citrate 3-4 101 78-125 1-1 104 94-117 Plasma 34-35 101 75-126 13-13 111 97-120 (N = 10) 321-361 98 79-117 126-127 115 88-130 Urine 3-4 104 99-111 1-1 110 100-117  (N = 5) 32-35 108 105-114  13-13 111 97-125 305-321 114 109-125  125-127 120 108-135  Cell Culture 9 107 97-119 6 84 71-93  Supernates 86 99 90-105 66 80 74-86  (N = 6) 838 106 88-130 691 94 77-93  IL-12/IL-23 p40 IL-15 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 21-21 92 81-104 3-4 98  78-110 (N = 11) 194-221 90 79-101 35-36 100  76-118 1819-2112 94 78-107 284-300 119  92-138 EDTA 21-21 91 81-99  4-4 100  92-110 Plasma 194-199 97 88-109 35-35 108  97-120 (N = 11) 1819-1951 96 79-130 290-300 122 103-143 Heparin 19-21 89 72-99  4-4 92  78-112 Plasma 193-199 86 71-121 34-35 103  88-132 (N = 11) 1684-1951 88 74-127 261-290 123 100-150 Citrate 19-20 91 85-99  4-4 104  87-113 Plasma 188-193 89 77-105 34-35 118 102-131 (N = 10) 1645-1684 95 71-117 261-278 150 129-173 Urine 20-20 127 119-131  3-4 105  98-113 (N = 5) 188-204 121 102-140  33-35 120 111-131 1645-1758 126 118-135  276-278 141 126-152 Cell Culture 38 119 104-132  6 76 71-89 Supernates 346 112 96-125 58 71 68-78 (N = 6) 3784 110 105-143  593 85 71-95 IL-16 IL-17A Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 17-17 90 80-102 7-7 102 82-132 (N = 11) 142-146 94 83-110 63-70 103 88-129 1594-1627 90 73-103 682-833 91 77-124 EDTA 17-17 88 82-99  7-7 97 81-110 Plasma 142-153 98 89-108 63-69 102 84-120 (N = 11) 1627-1865 96 85-116 682-721 95 80-120 Heparin 17-18 90 82-101 7-7 97 60-145 Plasma 153-155 92 83-107 69-72 97 72-115 (N = 11) 1665-1713 90 78-114 703-721 96 78-113 Citrate 18-18 89 84-97  7-9 96 56-113 Plasma 155-156 100 91-112 72-79 99 59-117 (N = 10) 1679-1713 104 83-124 667-703 97 49-113 Urine 18-19 78 65-96  8-9 131 119-142  (N = 5) 153-156 75 59-87  74-79 142 126-171  1673-1679 68 48-83  667-711 134 105-155  Cell Culture 31 102 87-131 53 111 91-119 Supernates 287 85 78-96  561 117 96-128 (N = 6) 3311 101 83-110 6298 122 108-133  TNFβ VEGF Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 9-9 88 76-103 10-11 81 61-96  (N = 11) 85-93 84 69-104 112-119 73 37-103 882-884 85 63-108 1708-1733 65 40-104 EDTA 9-9 93 71-105 11-12 88 80-115 Plasma 85-91 92 66-105 119-120 91 82-110 (N = 11) 884-904 89 67-110 1706-1733 88 74-101 Heparin 8-9 89 64-128 12-17 78 39-96  Plasma 85-91 89 69-127 120-142 62 39-118 (N = 11) 798-904 88 66-124 1676-1706 47 32-110 Citrate 8-8 70 44-90  17-19 71 35-96  Plasma 85-85 87 42-81  142-158 83 67-121 (N = 10) 784-798 69 44-87  1676-1739 78 58-113 Urine 8-8 135 123-148  18-19 94 88-98  (N = 5) 83-85 138 128-152  153-158 118 107-134  784-816 139 128-149  1739-1749 119 111-124  Cell Culture 6 117 91-132 12 92 85-100 Superntates 65 111 89-126 118 93 88-102 (N = 6) 644 119 88-130 1718 140 131-151 

TABLE 25 Panel 3 Eotaxin MIP-1β Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 70-75 100 86-113 51-54 107 72-123 (N = 12) 295-304 100 79-124 210-213 106 57-119 2336-2467 100 84-135 1988-2256 103 47-127 EDTA 74-75 99 95-114 54-59 106 68-120 Plasma 295-312 103 96-113 213-228 102 57-114 (N = 12) 2336-2483 113 96-129 1988-2114 100 56-119 Heparin 73-74 109 74-126 58-59 103 71-117 Plasma 312-331 91 68-106 228-241 97 51-109 (N = 12) 2345-2483 79 64-104 1866-2114 99 50-116 Citrate 73-73 100 82-113 58-59 108 102-121  Plasma 331-338 95 75-103 241-263 102 92-109 (N = 10) 2345-2483 99 76-119 1866-2203 107 93-121 Urine 73-73 111 97-119 56-59 114 109-121  (N = 5) 331-338 104 94-116 250-263 104 96-109 2398-2483 101 75-125 2016-2203 102 89-115 Cell Culture 70-75 100 86-113 51-54 107 72-123 Supernates 295-304 100 79-124 210-213 106 57-119 (N = 6) 2336-2467 100 84-135 1988-2256 103 47-127 Eotaxin-3 TARC Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 250-271 105 83-135 65-74 106 71-123 (N = 12) 1044-1189 100 78-135 269-270 112 78-133 12 348-12 607 106 87-164 2719-2753 120 99-153 EDTA 271-279 126 114-135  74-79 96 69-105 Plasma 1044-1116 135 119-150  269-279 105 79-119 (N = 12) 12 348-13 154 145 102-177  2719-2969 106 77-127 Heparin 279-284 106 90-129 70-79 104 44-140 Plasma 1116-1257 106 92-128 279-287 87 46-111 (N = 12) 11 390-13 154 130 85-171 2592-2969 91 59-111 Citrate 273-284 110 93-126 70-70 105 94-121 Plasma 1257-1310 104 93-114 287-308 108 94-123 (N = 10) 11 390-11 682 112 98-130 2592-2692 124 100-226  Urine 273-298 113 109-119  70-82 129 98-162 (N = 5) 1310-1356 96 92-100 308-337 115 83-141 11 682-11 892 96 89-103 2692-2742 112 83-140 Cell Culture 262-297 91 84-95  81-89 119 115-126  Supernates 1161-1276 94 90-99  358-375 120 117-123  (N = 6)  9226-10 407 101 95-108 3194-3905 125 110-135  IP-10 MIP-1α Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 126-130 101 91-110 52-55 105 88-117 (N = 12) 475-522 111 102-120  213-215 110 90-125 5723-6188 113 79-131 1922-2000 112 91-123 EDTA 126-138 104 99-113 55-56 103 85-113 Plasma 475-511 115 103-124  213-218 112 95-125 (N = 12) 5982-6188 112 91-134 1922-2009 114 92-123 Heparin 138-145 99 92-104 55-56 97 80-106 Plasma 511-563 106 96-114 218-241 103 77-115 (N = 12) 5982-6172 117 77-138 1781-2009 113 77-130 Citrate 144-145 103 91-121 55-55 105 65-141 Plasma 563-632 102 87-113 241-242 104 67-115 (N = 10) 6172-6601 110 88-139 1781-1916 119 78-133 Urine 144-153 111 104-124  54-55 116 104-127  (N = 5) 566-632 102 97-108 242-242 115 109-123  6492-6601 84 55-97  1916-2167 121 116-129  Cell Culture 116-144 110 97-121 44-52 112 100-118  Supernates 548-681 134 121-150  230-259 117 111-124  (N = 6)  5276-28 154 274 113-603  2341-2692 119 110-127  IL-8 MCP-1 Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 3828-3911 87 76-97  22-24 97 87-107 (N = 12) 12 490-12 841 91 81-110 91-92 102 90-109 156 805-157 779 88 77-99  916-928 100 77-109 EDTA 3911-4338 94 86-100 24-26 88 77-99  Plasma 12 490-13 720 104 81-115 91-94 90 84-96  (N = 12) 153 349-156 805 94 74-110 928-972 85 72-102 Heparin 3881-4338 84 73-93  26-27 102 88-130 Plasma 13 720-14 211 88 78-97   94-103 105 94-123 (N = 12) 143 226-153 349 93 71-102 877-972 100 80-116 Citrate 3727-3881 98 78-109 26-27 94 85-108 Plasma 14 211-14 529 94 82-102 103-108 95 85-108 (N = 10) 143 226-144 105 102 92-115 877-954 92 81-110 Urine 3727-3913 114 108-119  26-27 103 95-116 (N = 5) 13 811-14 529 105 101-108  106-108 106 96-117 144 105-159 737 105 103-106  924-954 102 90-112 Cell Culture 3697-4844 86 71-93  21-24 101 96-109 Supernates 17 099-19 472 103 95-109  98-105 106 103-110  (N = 6) 167 913-183 991 103 97-106  994-1187 106 99-119 MDC MCP-4 Spike Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 421-443 97  86-115 31-33 104 70-124 (N = 12) 1707-1780 122 113-129 123-133 94 57-108 23 795-24 625 120 115-127 1016-1031 95 71-104 EDTA 443-465 99  91-105 31-32 107 78-127 Plasma 1707-1744 115 107-123 123-136 90 57-106 (N = 12) 23 795-26 159 113 101-128 1016-1085 89 64-101 Heparin 439-465 104  94-117 32-32 128 113-146  Plasma 1744-1948 114 101-130 136-137 108 86-121 (N = 12) 21 657-26 159 105  92-119  960-1085 94 72-111 Citrate 426-439 105  93-126 31-32 118 104-148  Plasma 1948-2048 111 106-125 137-147 97 85-107 (N = 10) 21 657-23 766 117  99-151  960-1075 102 90-111 Urine 426-465 130 112-140 30-31 125 118-134  (N = 5) 1967-2048 123  96-138 146-147 107 96-115 22 796-23 766 124  99-140 1057-1075 107 101-118  Cell Culture 527-592 129 122-137 16-24 87 64-93  Supernates 2694-2954 142 138-152 125-132 98 95-100 (N = 6) 33 418-39 552 147 134-159 1120-1201 103 100-107 

TABLE 26 Panel 4 IFNγ IL-2 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Seram 833.3 92%  84%-102% 8333.3 90% 79%-101% (N = 6) 208.3 88% 82%-96% 2083.3 98% 81%-108% 52.1 89% 80%-96% 520.8 102%  82%-113% EDTA 833.3 89% 86%-99% 8333.3 98% 89%-106% Plasma 208.3 89% 68%-98% 2083.3 104%  96%-110% (N = 6) 52.1 94%  86%-101% 520.8 109%  102%-115%  Heparin 833.3 71% 66%-75% 8333.3 98% 91%-106% Plasma 208.3 70% 68%-71% 2083.3 103%  99%-105% (N = 6) 52.1 63% 57%-68% 520.8 111%  104%-118%  Urine 833.3 88% 76%-96% 8333.3 64% 42%-86%  (N = 6) 208.3 93%  74%-103% 2083.3 65% 43%-85%  52.1 100%   88%-108% 520.8 71% 42%-92%  Cell Culture Supernates (N = 4) IL-4 IL-1β Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 170 96%  91%-102% 1800 59% 51%-65% (N = 6) 42.5 84% 80%-85% 450 66% 62%-72% 10.6 91% 90%-94% 112.5 77% 62%-93% EDTA 170 102%   99%-107% 1800 69% 65%-72% Plasma 42.5 94%  91%-104% 450 80% 74%-86% (N = 6) 10.6 103%   97%-108% 112.5 87% 82%-94% Heparin 170 91% 82%-95% 1800 76% 69%-81% Plasma 42.5 80% 69%-90% 450 82% 73%-88% (N = 6) 10.6 87% 74%-98% 112.5 87%  77%-104% Urine 170 75%  43%-101% 1800 265%  166%-389% (N = 6) 42.5 65% 41%-85% 450 183%  102%-253% 10.6 71% 47%-85% 112.5 161%   91%-235% Cell Culture Supernates (N = 4) IL-5 IL-6 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 1666.7 87% 80%-97%  2066.7  95%  83%-108% (N = 6) 416.7 88% 82%-90%  516.7 107%  83%-116% 129.2 83% 67%-100% 129.2 109%  89%-127% EDTA 1666.7 81% 69%-91%  2066.7  97%  88%-111% Plasma 416.7 84% 71%-93%  516.7 113%  98%-139% (N = 6) 129.2 91% 81%-100% 129.2 126% 111%-135% Heparin 1666.7 91% 81%-110% 2066.7 106%  99%-112% Plasma 416.7 92% 81%-114% 516.7 117% 105%-130% (N = 6) 129.2 81% 58%-92%  129.2 128% 111%-154% Urine 1666.7 77% 32%-132% 2066.7 130% 124%-138% (N = 6) 416.7 80% 44%-118% 516.7 141% 126%-165% 129.2 90% 52%-140% 129.2 147% 118%-201% Cell Culture Supernates (N = 4) KC/GRO IL-10 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 463.3 84% 68%-96% 3333.3 91%  80%-102% (N = 6) 115.8 90% 87%-98% 833.3 94% 83%-99% 29 99%  94%-109% 208.3 99%  86%-112% EDTA 463.3 88% 76%-95% 3333.3 98%  86%-106% Plasma 115.8 92%  78%-101% 833.3 113%  104%-125% (N = 6) 29 122%   94%-134% 208.3 117%  110%-130% Heparin 463.3 59% 41%-81% 3333.3 82% 66%-96% Plasma 115.8 61% 42%-77% 833.3 79% 70%-88% (N = 6) 29 76% 51%-99% 208.3 75% 67%-83% Urine 463.3 142%  132%-170% 3333.3 128%  118%-135% (N = 6) 115.8 130%  119%-157% 833.3 119%  104%-139% 29 120%  107%-135% 208.3 119%  100%-136% Cell Culture Supernates (N = 4) IL-13 TNFα Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum 266.7 80% 66%-88% 193.3 87% 81%-92% (N = 6) 66.7 71% 67%-75% 48.3 83% 79%-87% 16.7 77% 65%-87% 12.1 100%   93%-112% EDTA 266.7 92% 89%-95% 193.3 86% 82%-93% Plasma 66.7 89% 86%-95% 48.3 84% 79%-91% (N = 6) 16.7 105%  102%-114% 12.1 91% 87%-96% Heparin 266.7 82% 73%-90% 193.3 81% 72%-85% Plasma 66.7 63% 58%-68% 48.3 73% 69%-77% (N = 6) 16.7 70% 58%-92% 12.1 84%  66%-107% Urine 266.7 82%  83%-121% 193.3 82% 46%-97% (N = 6) 66.7 83%  74%-110% 48.3 83% 52%-92% 16.7 104%   86%-121% 12.1 104%   62%-111% Cell Culture Supernates (N = 4)

TABLE 27 Panel 5 IFNγ IL-1β Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum High (390-580) 117 100-128  High (1080-1850) 106 94-118 (N = 8) Mid (90-370) 117 91-137 Mid (380-530) 106 81-121 Low (8-90) 112 96-123 Low (10-180) 103 84-112 EDTA High (420-580) 117 97-142 High (1120-1990) 114 94-135 Plasma Mid (90-330) 110 103-120  Mid (420-570) 105 98-117 (N = 7) Low (80-90) 83 85-100 Low (140-150) 95 90-100 Heparin High (380-580) 105 95-109 High (1120-1730) 98 87-104 Plasma Mid (80-330) 105 98-114 Mid (380-570) 107 95-117 (N = 7) Low (90-90) 87 89-100 Low (140-150) 94 89-102 Citrate High (380-580) 115 100-131  High (1080-1800) 99 93-108 Plasma Mid (80-370) 110 93-120 Mid (400-530) 101 85-121 (N = 8) Low (8-90) 102 92-109 Low (20-180) 105 85-123 Urine High (450-590) 102 91-117 High (1120-1830) 89 82-90  (N = 5) Mid (90-340) 105 99-110 Mid (260-590) 89 87-91  Low (20-90) 105 97-117 Low (70-150) 94 99-105 Cell Culture High (500-680) 102 96-111 High (950-1130) 100 93-108 Supernates Mid (280-330) 111 110-112  Mid (540-570) 104 102-106  (N = 4) Low (70-90) 105 104-108  Low (140-150) 101 99-104 IL-2 IL-4 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum High (1540-2150) 108 94-115 High (990-1430) 74 59-86 (N = 8) Mid (470-870) 105 94-117 Mid (350-600) 68 53-77 Low (10-220) 105 94-113 Low (10-150) 71 53-81 EDTA High (1580-2400) 108 103-117  High (1030-1550) 67 58-81 Plasma Mid (490-850) 105 99-109 Mid (330-530) 68 58-72 (N = 7) Low (200-240) 108 100-111  Low (140-140) 67 59-72 Heparin High (1580-2090) 97 91-102 High (1030-1370) 56 48-80 Plasma Mid (470-850) 97 93-103 Mid (310-530) 59 57-81 (N = 7) Low (200-240) 98 90-104 Low (140-140) 59 54-87 Citrate High (1540-2220) 100 90-111 High (990-1320) 68 64-73 Plasma Mid (500-870) 99 95-108 Mid (340-600) 65 58-72 (N = 8) Low (10-220) 99 92-110 Low (20-150) 66 55-75 Urine High (1670-2440) 91 83-100 High (1100-1580) 57 40-65 (N = 5) Mid (450-900) 89 80-99  Mid (310-580) 59 52-88 Low (90-220) 95 85-101 Low (80-140) 63 53-71 Cell Culture High (1480-1690) 103 98-109 High (820-1040) 96  91-105 Supernates Mid (820-850) 108 105-111  Mid (520-530) 104 103-105 (N = 4) Low (200-210) 108 107-109  Low (130-140) 99  99-100 IL-5 IL-6 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum High (650-950) 111 101-121  High (3330-4680) 112 88-131 (N = 8) Mid (230-380) 108 92-122 Mid (900-1980) 105 72-122 Low (8-90) 103 95-107 Low (72-540) 105 82-118 EDTA High (570-1370) 106 83-128 High (3510-5400) 117 85-143 Plasma Mid (320-330) 102 92-117 Mid (900-1800) 108 86-118 (N = 7) Low (80-90) 98 92-104 Low (450-3870) 89 84-97  Heparin High (870-990) 96 79-118 High (3510-4590) 99 80-115 Plasma Mid (230-330) 102 87-114 Mid (810-1800) 104 86-116 (N = 7) Low (80-90) 98 85-108 Low (450-420) 88 82-92  Citrate High (860-1130) 104 90-115 High (3330-4410) 102 91-113 Plasma Mid (250-380) 103 91-114 Mid (990-1980) 98 81-115 (N = 8) Low (10-90) 97 81-113 Low (90-540) 97 78-108 Urine High (710-1010) 94 85-107 High (3780-4880) 88 80-90  (N = 5) Mid (240-370) 94 89-100 Mid (810-1980) 90 72-94  Low (40-90) 100 87-119 Low (100-540) 90 73-108 Cell Culture High (590-680) 103 100-107  High (3050-3530) 109 103-114  Supernates Mid (290-330) 111 108-114  Mid (170-1800) 111 108-113  (N = 4) Low (80-90) 99 93-104 Low (430-460) 109 105-111  KC/GRO IL-10 Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum High (1150-1920) 84 79-90 High (2180-6020) 87 80-96 (N = 8) Mid (380-640) 82 74-91 Mid (1080-1230) 87 72-98 Low (30-160) 85 73-89 Low (40-310) 89 75-99 EDTA High (1330-1940) 101  82-113 High (2280-4840) 103  88-130 Plasma Mid (350-630) 99  87-107 Mid (970-1150) 98  91-105 (N = 7) Low (150-150) 93  85-101 Low (270-280) 101  95-108 Heparin High (1330-1730) 107  90-129 High (2280-4810) 85 73-95 Plasma Mid (380-630) 95  91-100 Mid (1010-1150) 91 86-95 (N = 7) Low (140-150) 97  89-103 Low (240-280) 98  89-111 Citrate High (1150-1730) 112  99-123 High (2180-4880) 90 82-96 Plasma Mid (370-640) 111  98-121 Mid (1130-1230) 93  84-100 (N = 8) Low (50-160) 100  91-112 Low (30-310) 89 77-97 Urine High (1350-1930) 131 116-142 High (2170-5290) 94 84-99 (N = 5) Mid (310-600) 135 123-145 Mid (1020-1130) 95  90-101 Low (80-100) 120 122-164 Low (200-290) 99  90-106 Cell Culture High (1010-1340) 125 122-129 High (1690-2270) 112 100-128 Supernates Mid (610-630) 135 125-141 Mid (1010-1150) 121 118-126 (N = 4) Low (140-150) 132 122-138 Low (240-280) 125 122-127 IL-12p70 INFα Spike Level & Spike Level & Sample Conc. Range Average % % Recovery Conc. Range Average % % Recovery Type (pg/mL) Recovery Range (pg/mL) Recovery Range Serum High (20 200-30 710) 128 119-144 High (391-510) 90 85-96 (N = 8) Mid (6220-11640) 126 107-139 Mid (85-220) 92  75-101 Low (1110-2550) 118 111-124 Low (17-88) 92 82-95 EDTA High (21 960-26 790) 152 115-194 High (425-544) 95  89-113 Plasma Mid (8240-10880) 123 116-128 Mid (102-221) 95  94-105 (N = 7) Low (2380-2580) 118 111-124 Low (51-102) 91 88-95 Heparin High (21 960-28 320) 133 118-143 High (425-493) 88 82-93 Plasma Mid (5830-10850) 117 108-127 Mid (85-221) 95 91-99 (N = 7) Low (2380-2410) 100  91-105 Low (51-102) 91 87-95 Citrate High (20 200-28 280) 127 100-196 High (391-478) 94  87-106 Plasma Mid (6370-11640) 121  85-187 Mid (102-238) 94 91-96 (N = 8) Low (1110-2550) 114  86-143 Low (34-88) 96  88-100 Urine High (20900-30370) 90 79-97 High (425-527) 74 69-84 (N = 5) Mid (5510-10710) 91 85-98 Mid (102-238) 77 67-87 Low (1040-2430) 102  86-124 Low (17-88) 83 71-95 Cell Culture High (18990-21960) 116 112-123 High (350-420) 113 105-121 Supernates Mid (10830-10880) 120 116-123 Mid (190-210) 117 113-121 (N = 4) Low (2380-2400) 122 119-125 Low (50-60) 112 108-116

To assess specificity of the individual assays, each panel was run using blended antibodies with individual calibrators at concentration that yield signal around 100,000 counts.

${{Non}\text{-}{{specificity}(\%)}} = {\left( \frac{{Specific}\mspace{14mu} {Signal}}{{Non}\text{-}{specific}\mspace{14mu} {Signal}} \right)*100}$

TABLE 28 Panel 1 Calibrator IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Concentration 153 34 92 40 120 50 100 313 208 120 (pg/mL) Highest non- 0.01 0.01 0.03 0.02 0.51 0.15 0.01 0.01 0.05 0.02 specificity (%)

TABLE 29 Panel 2 Calibrator GM-CSF IL-1α IL-5 IL-7 IL-12/IL-23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Concentration 125 30 33 82 328 88 469 457 43 108 (pg/mL) Highest non- 0.13 0.11 0.08 0.05 0.22 0.81 0.10 0.12 0.89 0.08 specificity (%)

TABLE 30 Panel 3 Calibrator Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Concentration 250 100 1250 100 200 150 6000 100 300 156 (pg/mL) Highest non- 0.02 0.06 0.03 0.04 0.33 0.63 0.03 0.04 0.04 0.05 specificity (%)

TABLE 31 Panel 4 Calibrator IFNγ IL-2 IL-4 IL-1β IL-5 IL-6 KC/GRO IL-10 IL-13 TNFα Concentration 1250 15 000 300 13 000 2000 3830 600 3830 750 200 (pg/mL) Highest Non- 0.05    0.08 0.08    0.00 0.14 0.25 0.34 0.47 0.81 0.81 specificity (%)

TABLE 32 Panel 5 Calibrator IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα Concentration 40 100 160 100 60 420 200 500 5020 83 (pg/mL) Highest Non- 0.01 0.03 0.02 0.02 0.04 0.19 0.19 0.46 0.07 0.03 specificity (%)

To assess the specificity of each antibody, each panel was run using blended calibrators with concentrations listed above and individual antibodies at 1× concentration.

TABLE 33 Panel 1 Antibody IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Highest non- 0.02 0.02 0.03 0.02 0.52 0.11 0.05 0.11 0.04 0.43 specificity (%)

TABLE 34 Panel 2 Antibody IL-12/IL-23 GM-CSF IL-1α IL-5 IL-7 p40 IL-15 IL-16 IL-17A TNFα VEGF Highest non- 0.08 0.10 0.05 0.06 0.19 0.27 0.47 0.12 0.06 0.02 specificity (%)

TABLE 35 Panel 3 Antibody Eotaxin MP-1β Eotaxin-3 TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Highest non- 0.05 0.29 0.20 0.12 0.36 0.34 0.49 0.35 1.2 specificity (%)

TABLE 36 Panel 4 Antibody IFNγ IL-2 IL-4 IL-1β IL-5 IL-6 KC/GRO IL-10 IL-13 TNFα Highest Non- 0.00 0.1 0.08 0.07 0.07 0.12 0.22 0.11 0.25 0.08 specificity (%)

TABLE 37 Panel 5 Antibody IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα Highest Non- 0.01 0.00 0.01 0.05 0.02 0.02 0.05 0.04 0.03 0.04 specificity (%)

To evaluate the specificity of the Panel 1 Kit assays against other biomarkers, each kit was run using blended antibodies with individual recombinant human proteins.

TABLE 38 Panel 1 Protein IL-12/IL-23 IL-5 GM-CSF IL-1α IL-7 p40 IL-15 IL-16 IL-17A TNFβ VEGF Concentration 49 63 23 47 188 44 156 304 38 67 (pg/mL) Highest non- 0.15 0.28 1 0.25 0.43 0.42 0.78 0.29 0.16 0.91 specificity (%) Protein Eotaxin MP-1β Eotaxin-3 TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Concentration 94 63 313 94 156 62 31 625 39 (pg/mL) Highest non- 0.71 0.48 0.45 0.17 0.24 1 0.57 0.27 1 specificity (%)

TABLE 39 Panel 2 Protein IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-13 TNFα Concentration 78 31 78 13 41 4 19 29 21 (pg/mL) Highest non- 0.31 0.23 0.16 0.23 0.26 0.26 0.27 0.68 0.37 specificity (%) Protein Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α MCP-1 MDC MCP-4 Concentration 94 63 313 94 156 62 31 625 39 (pg/mL) Highest non- 0.3 0.57 0.32 0.14 0.11 0.85 0.25 0.19 0.92 specificity (%)

TABLE 40 Panel 3 Protein IL-5 GM-CSF IL-1α IL-7 IL-12/IL-23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Concentration 49 63 23 47 100 44 156 304 30 67 (pg/mL) Highest non- 0.19 0.10 0.34 0.22 0.26 0.30 0.49 0.10 0.10 0.41 specificity (%) Protein IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Concentration 70 31 70 13 41 4 19 24 29 21 (pg/mL) Highest non- 0.28 0.09 0.10 0.10 0.19 N/A 0.39 0.61 0.33 0.23 specificity (%) To evaluate the impact of multiplexing on assay signal, standards in the quantifiable ranges were compared between individual assays (individual calibrator and individual antibody) and multiplexed assays (blended calibrators and blended antibodies) using each kit. The calculated % signal difference between individual and multiplexed assay is shown below.

TABLE 41 Panel 1 Assay IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Signal 0.02 0.02 0.03 0.02 0.52 0.11 0.05 0.11 0.04 0.43 Difference (%)

TABLE 42 Panel 2 Assay IL-12/IL-23 GM-CSF IL-1α IL-5 IL-7 p40 IL-15 IL-16 IL-17A TNFβ VEGF Signal 4 30 24 27 2 8 22 14 3 10 Difference (%)

TABLE 43 Panel 3 Assay Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Signal 4 4 24 11 22 2 7 3 6 11 Difference (%)

TABLE 44 Panel 4 Antibody IFNγ IL-2 IL-4 IL-1β IL-5 IL-6 KC/GRO IL-10 IL-15 TNFα Signal 11 17 13 23 37 29 3 3 5 9 Difference (%)

TABLE 45 Panel 5 Assay IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα Signal 14 10 2 16 15 8 7 0 1 17 Difference (%)

The kits were designed to minimize interference by receptors and other related proteins. For each panel, a multi-analyte calibrator in diluent and normal human were spiked with three different concentrations of receptors and binding partners. The recovered calibrator concentrations were compared to unspiked standards and normal serum.

All the assays in each panel were calibrated against a reference calibrator obtained from Meso Scale Discovery (Rockville, Md.). The NIBSC/WHO Standards for the following human analytes were evaluated against the MSD reference calibrators. To convert sample values obtained with a panel to approximate NIBSC/WHO concentration, the calculated sample value was multiplied by the concentration ratio.

TABLE 46 Panel 1 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference: NIBSC) IL-1β 86/680 1.0 IL-2 86/504 1.1 IL-4 88/656 1.0 IL-6 89/548 1.0 IL-8 89/520 1.0 IL-10 93/322 1.0 IL-12p70 95/544 1.0 IL-13 94/622 1.0 TNFα 88/186 1.0

TABLE 47 Panel 2 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference: NIBSC) GM-CSF 88/646 1.08 IL-1α 86/632 1.0 IL-5 90/586 1.0 IL-7 90/530 1.0 IL-15 95/554 0.95 IL-17A 01/420 1.0 TNFα 87/640 1.0 VEGF 02/286 1.0

TABLE 48 Panel 3 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference: NIBSC) MIP-1α 92/518 1.0 IL-8 89/520 1.0 MCP-1 92/394 0.85

TABLE 49 Panel 5 Concentration Ratio Analyte NIBSC/WHO Standard (MSD Reference: NIBSC) IL-1β 96/668 1.18 IL-2 93/566 0.98 IL-4 91/656 0.89 IL-6 93/730 1.0 TNFα 88/532 1.0

(a) Normal Sample Testing

Normal mouse serum (rat serum for panel 4), EDTA plasma, heparin plasma, citrate plasma, and urine samples from a commercial source were diluted 2- to 4-fold and tested with each panel. Median and range of concentrations for each sample set are displayed below. Concentrations are corrected for sample dilution.

TABLE 50 Panel 1 Sample Type Statistic IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Serum Median 3.77 0.0955 0.403 0.00565 0.167 9.61 0.0605 0.0102 0.0994 0.199 (N = (pg/mL) 27) Range 1-14 0-14 0-3 0-0  0-27   1-1,721 0-3 0-0 0-3 0-2 (pg/mL) Samples 26 12 15 14 17 27 26 16 12 27 in Quan- titative Range EDTA Median 3.80 0.538 0.174 0.0166 0.174 0.519 0.167 0.150 0.0 0.735 Plasma (pg/mL) (N = Range 0-23 0-1  0-4 0-0 0-1 0-20  0-3 0-1 0-1 0-2 22) (pg/mL) Samples 21 22 16 17 15 22 21 17 13 22 in Quan- titative Range Heparin Median 2.87 0.0894 0.0510 0.0 0.114 60.1 0.0798 0.0473 0.0 0.456 Plasma (pg/mL) (N = Range 0-8  0-11 0-3 0-0 0-3  2-2626 0-3 0-0 0-3 0-1 27) (pg/mL) Samples 27 27 16 13 18 27 23 15 17 27 in Quan- titative Range Citrate Median 3.28 0.0627 0.0897 0.00496 0.190 2.85 0.115 0.0283 0.0 0.481 Plasma (pg/mL) (N = Range 1-35 0-0  0-1 0-0 0-0 0-112 0-2 0-0 0-0 0-3 20) (pg/mL) Samples 20 11 10 12 10 20 20 11 10 20 in Quan- titative Range Urine Median 0.399 0.350 0.0513 0.0168 0.088 35.185 0.018 0.009 0.0 0.0 (N = (pg/mL) 5) Range 0-1  0-10 0-0 0-0 0-0 1-105 0-0 0-0 0-0 0-0 (pg/mL) Samples 5 5 5 5 5 5 5 5 5 5 in Quan- titative Range ND = Non-detectable

TABLE 51 Panel 2 Sample IL-12/IL- Type Statistic GM-CSF IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Serum Median  34.1 1 ND 1 52 1 60 5 ND  9 (N = (pg/mL) 20) Range 11-44 1-62 ND 1-3 13-159 1-3 24-137 5-5 ND 2-187 (pg/mL) Samples 8 9 0 15  20 20  20 1 0 15 in Quan- titative Range EDTA Median   1.5 2 1 3 65 2 76 9 ND 95 Plasma (pg/mL) (N = Range 0-1 0-99 1-1   0-40  3-395 1-3  0-973  1-55 ND 18-338  20) (pg/mL) Samples 2 18  1 15  17 16  15 5 0 13 in Quan- titative Range Heparin Median ND 1 45  100  ND 1  5 1 ND  9 Plasma (pg/mL) (N = Range ND 1-1  9-148  35-1109 ND  1-57 1-39 1-2 ND 5-484 20) (pg/mL) Samples 0 2 19  19   0 7 17 19  0 17 in Quan- titative Range Citrate Median ND ND ND ND ND ND ND ND ND ND Plasma (pg/mL) (N = Range ND ND ND ND ND ND ND ND ND ND 20) (pg/mL) Samples 0 0 0 0  0 0  0 0 0  0 in Quan- titative Range Urine Median 2 3 ND 1 27 1 47 5 ND 50 (N = (pg/mL) 5) Range 2-2 2-3  ND 1-1 27-27  1-2 47-47  5-5 ND 37-85  (pg/mL) Samples 1 3 0 2  1 2  1 1 0  4 in Quan- titative Range ND = Non-detectable

TABLE 52 Panel 3 Sample Type Statistic Eotaxin MSP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Serum Median 41 46  6 28 65  9 547 107  1,246   34 (N = (pg/mL) 27) Range 19-145 7-95 5-9  5-70 27-261 7-202 262-665  76-205 606-3249 11-117 (pg/mL) Samples 26 27 10 27 27 19  7 27 27 27 in Quan- titative Range EDTA Median 117  63 13 79 197  11 769 79 1,309   67 Plasma (pg/mL) (N = Range 37-795  8-153 5-115 13-373 97-876 7-651 340-2478 42-185 869-2144 12-582 23) (pg/mL) Samples 27 27 26 27 27 20  11 27 27 27 in Quan- titative Range Heparin Median 293  121  31 92 146  41 775 137  1,050   183  Plasma (pg/mL) (N = Range  22-1522 10-301 5-147  6-957 91-625  7-2231 234-3281 69-319 589-1963 67-716 27) (pg/mL) Samples 27 27 27 27 27 26  7 27 27 27 in Quan- titative Range Citrate Median 191  51 12 51 77  9 964 135  994  64 Plasma (pg/mL) (N = Range 72-288 19-123 7-19  25-131 36-373 7-30  328-1869 76-242 576-1364 35-161 20) (pg/mL) Samples 20 20 19 20 20  9  4 20 20 20 in Quan- titative Range Urine Median 13  3 ND  1  8 ND 296 80 ND 13 (N = (pg/mL) 5) Range 13-13  2-13 ND 1-1  3-64 ND 296-296  56-122 ND 11-15  (pg/mL) Samples  1  5  0  1  3  0  1  5  0  2 in Quan- titative Range ND = Non-defectable

TABLE 53 Panel 5 Sample Type Statistic IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 INFα Serum Median 0.95 2.27 1.02 0.43 2.32 21.6 48.3 11.0 81.0 12.0 (N = (pg/mL) 16) Range 0.34-28.3 1.13-3.95 0.55-3.98 0.23-1.10 0.58-6.52 5.28-110.3  28.3-101.8 5.71-45.4 64.8-97.1 8.23-34.4 (pg/mL) Samples 16 16 16 15 16 16 16 16 2 16 in Quan- titative Range EDTA Median 41.2 0.86 3.86 0.63 2.59 113.1 30.5 56.5 69.3 38.5 Plasma (pg/mL) (N = Range  18.6-262.1 0.46-2.40 2.60-5.89 0.48-0.70 1.50-2.88 11.0-184.6 54.2-96.9 31.5-74.7  50.2-170.8 21.3-47.0 15) (pg/mL) Samples 15 13 15 9 15 15 15 15 11 15 in Quan- titative Range Heparin Median 261.5 1.62 4.63 0.75 4.01 175.2 269.4 76.4 85.6 65.3 Plasma (pg/mL) (N = Range 156.4-352.4 0.61-2.25 3.35-7.36 0.42-1.49 2.26-5.32 28.8-354.6 220.1-368.6  63.7-104.6  38.0-152.0 35.0-76.7 15) (pg/mL) Samples 15 13 15 9 15 15 15 15 8 15 in Quan- titative Range Citrate Median 7.04 1.01 3.09 0.73 3.37 41.9 65.3 30.7 71.2 42.8 Plasma (pg/mL) (N = Range  0.31-121.8 0.45-2.02 0.65-5.03 0.39-1.43 1.32-8.24 6.84-34.2   34.9-132.0 5.30-68.2  50.4-107.4 5.45-58.8 16) (pg/mL) Samples 16 16 15 16 16 16 16 16 15 16 in Quan- titative Range Urine Median 0.32 0.57 0.49 0.43 ND ND 2.31 1.36 101.6 0.63 (N = (pg/mL) 10) Range 0.09-0.66 0.35-1.34 0.49-0.65 0.43-0.62 ND ND 1.91-2.84 0.98-1.53  67.3-125.1 0.48-3.90 (pg/mL) Samples 3 6 3 9 0 0 10 4 9 8 in Quan- titative Range ND = Non-detectable

(b) Stimulated Samples

Panel 1: Freshly collected normal human whole blood was incubated with LPS and simultaneously incubated with peptidoglycan (PG) and Zymosan (ZY) for different time periods and plasma was then isolated. These samples were then tested with panel 1. The dilution adjusted concentrations for each stimulation model is displayed below.

TABLE 54 Panel 1 Incubation Stimulant Time (hr) IFNγ IL-1β IL-2 IL4 IL-6 IL-8 IL-10 IL-12p70 IL-13 TNFα Control 0 11.8 0.22 0.68 0.09 0.63 6.08 0.37 0.37 ND 2.57 Control 6 hr 9.1 4.53 0.31 0.01 1.09 40.01 0.37 0.45 0.61 3.16 LPS 3 34.4 1028 ND 4.84 8028 5835 8.58 2.99 5.14 7962.8 12  458.6 9453 ND 0.11 22033 9778 115.8 4.14 ND 9158 PHA 100 μg 6 hr 0.04 14.51 ND ND ND 301.6 0.63 0.21 ND 9.30 PHA 1 μg 6 hr 14.7 184.5 ND 0.13 3669 1978.8 10.4 0.23 ND 261.18

Panel 2: Freshly collected normal human whole blood was incubated at 37° C. with LPS and PHA for different time periods and plasma was isolated. The samples were tested with panel 2.

TABLE 55 Panel 2 Incubation IL-12/IL- Stimulant Time (hr) GM-CSF IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNFβ VEGF Control 0 ND ND ND ND 96 2 73 ND ND ND Control 6 hr ND 12 ND 4 55 2 129 ND ND 44 LPS 3 ND ND ND ND 96 2 73 ND ND ND 12  ND 19 ND 3 6427 2 161 ND 1  3 PHA 100 μg 6 hr ND 10 ND 4 75 2 130 ND ND 33 PHA 1 μg 6 hr ND 16 ND 4 343 ND 794 ND ND 2096 

Panel 3: Freshly collected normal human whole blood was incubated at 37° C. with LPS for different time period and plasma was isolated. The samples were tested with panel 3.

TABLE 56 Panel 3 Incubation Stimulant Time (hr) Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Control 0 184 51 11 81 239 10 ND 114 1128 62 LPS (10 3 158 >4000 5 134 >10 000 >3960 3468 412 1891 89 ng/mL) 12 91 >4000 10 161 >10 000 >3960 2764 250 1210 114

Panel 5: Freshly collected normal pooled mouse whole blood was incubated with LPS and simultaneously incubated with peptidoglycan (PG) and Zymosan (ZY) for different time periods and plasma isolated. Samples were run on panel 5.

TABLE 57 Panel 5 Incubation Stimulant Time (hr) IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα None 0 11.76 1.02 2.74 0.50 3.20 135.40 96.75 34.02 26.17 35.40 (Control) LPS 3 6.00 7.49 1.85 8.73 6.73 1,510.01 66.75 57.20 115.60 200.37 12 11.73 89.00 1.63 10.90 6.92 3,352.90 120.59 47.35 130.06 360.90 None 3 6.00 2.59 2.58 4.60 2.49 400.00 99.27 31.55 93.99 30.65 (Control) PG/ZY 3 14.05 87.45 2.65 1.40 3.93 300.30 213.05 43.27 150.34 111.53 None 12 16.01 4.57 2.48 6.23 3.03 230.04 50.65 15.51 94.13 31.45 (Control) PG/ZY 12 30.76 150.00 3.01 3.03 3.00 1,500.23 159.85 59.44 90.93 723.63

For panels 1-3, freshly isolated PBMC from normal whole blood was stimulated with LPS, PHA, PWM, Con A, and co-stimulated with CD3 and CD28 antibodies. The samples were then tested with panels 1-3. The dilution adjusted concentrations in pg/mL for each stimulation model is displayed.

TABLE 58 Panel 1 Incubation Stimulant Time (hr) IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p7G IL-13 TNFα Control 24 hr 2.56 16.56 0.49 ND ND 4043 0.8 0.53 7.6 1.1 PWM 5 24 hr 13829 222 1614 20.28 9052 >1000 136.3 13.05 277.1 365.6 μg/mL LPS 10 24 hr 1494 >1000 ND ND 27701 >1000 748 9.8 228.5 >660 μg/mL PWM 50 24 hr 9468 5974 995 5.09 29858 205183 1123 3.96 5.5 1276 μg/mL A = CD3 + 24 hr 1747 4.11 571.2 11.03 ND 8017 42.9 ND 35 11.35 CD28 (5 + 5 μg/mL) Con A 20 24 hr 24658 116.5 3589 11.4 236.9 46528 146 24.04 45.08 263.5 μg/mL

TABLE 59 Panel 2 Incubation IL-12/IL- Stimulant Time (hr) GM-CSF IL-1α IL-5 IL-T 23 p40 IL-15 IL-16 IL-17A TNFα VEGF Control 24 hr ND ND ND 7 14 ND 1270 ND ND 1243 PWM 5 24 hr 178 51 291 14 338 ND 960 1677  9 1147 μg/mL LPS 10 24 hr 17 940 2 11 343 ND 794 ND ND 2096 μg/mL PWM 50 24 hr 66 811 31 9 526 ND 782 658  2 2194 μg/mL A = CD3 + 24 hr 78 339 10 132 ND 1046 321 27 804 CD28 (5 + 5 μg/mL) Con A 20 24 hr 212 272 194 11 1390 ND 1252 816 53 1299 μg/mL

TABLE 60 Panel 3 Incubation Stimulant Time (hr) Eotaxin MIP-1β Eotaxin-3 TARC IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Control  6 hr 140 181 33 132 312 41 ND 80 1157 47 Control 24 hr 127 76 31 199 2322 34 3742 141 769 59 PWM (100  6 hr 197 1753 31 147 372 117 NaN 71 942 55 mg/mL) PWM (1  6 hr 149 >4100 21 141 1653 1853 NaN 443 940 55 μg/mL) PWM (5 24 hr 130 19163 NaN 683 107930 10374 192588 80059 1986 278 μg/mL) LPS (10 24 hr 84 59243 NaN 258 3036 48161 182842 355 515 149 μg/mL) PWM (50 24 hr 131 59467 NaN 232 4169 43588 195681 465 560 123 μg/mL) CD3 + 24 hr 143 1358 NaN 1250 75234 534 4646 6844 2543 507 CD28 (5 μg/mL each) CON A (20 24 hr 230 4347 NaN 1307 105145 700 35480 43377 3889 320 μg/mL)

For panels 1-3, human acute monocyte leukemia cell line (THP-1 cell line) was stimulated with LPS for six and 16 hours. The supernates were then isolated and tested with panels 1-3. The dilution adjusted concentrations in pg/mL for each sample is displayed below.

TABLE 61 Panel 1 Incubation Stimulant Time (hr) IFNγ IL-1β IL-2 IL-4 IL-6 IL-8 IL-10 IL-12p70 I-15 TNFα Control 0 hr 1.09 20.48 0.345 0.07 0.19 449.8 0.44 0.38 1.41 9.91 LPS 6 hr 1.1 645.5 11.5 ND ND 61066 97.2 11.12 0.87 12472 16 hr  0.67 423 ND ND ND 69638 15.5 ND 1.06 2915

TABLE 62 Panel 2 Incubation IL-12/IL- Stimulant Time (hr) GM-CSF IL-1α IL-5 IL-7 23 p40 IL-15 IL-16 IL-17A TNFα VEGF Control 0 hr ND ND ND ND ND ND 205 ND ND 1995 LPS 6 hr ND 47 ND ND 56 ND 421 ND ND 276 16 hr  ND 22 ND ND 234 ND 552 ND ND >1070

TABLE 63 Panel 3 Incubation Stimulant Time (hr) Eotaxin MIP-1β Eotaxin-3 TAR IP-10 MIP-1α IL-8 MCP-1 MDC MCP-4 Control 0 hr 26 983 12 ND 96 70 1132 205 148 ND LPS 6 hr ND >4000 ND ND 324 >3960 45 546 577  14 998 57 16 hr  ND >4000 ND 20 687 2759 52 262 1290 >40 000 332

For panel 5, a mouse monocyte macrophage cell line (J774A.1) and a mouse leukemic monocyte macrophage cell line (RAW 264.7) were stimulated with different stimulants. The J774A.1 cell line stimulation was for four house while the RAW cell line stimulation was for six hours. The lysates were collected and run on panel 5. The concentrations are listed in pg/mL and normalized for 50 μg of lysate per well.

TABLE 64 Panel 5 Cell Lines Stimulant IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 KC/GRO IL-10 IL-12p70 TNFα J774A.1 None ND 1.9 ND ND ND 13 ND 34 ND 812 J774A.1 5 μg/mL LPS ND 8948 3.9 ND ND 62 529 107 320 ND >10 000 J774A.1 5 μ/mL PWM ND 10 674 2.8 ND ND 43 527 111 209 ND >10 000 J774A.1 1 ng/mL LPS ND 304 ND ND ND 403  074 57 ND 384 J774A.2 100 ng/mL PWM ND 150 ND ND ND 57 ND 54 ND 264 RAN 264.7 100 ng/mL LPS ND 41 458 1.0 0.16 ND 8129   12 1257 ND >10 000

The following calibrator blends were used in each panel as follows:

TABLE 65 Panel 1 Calibrator Sequence Expression System IFNγ Gln24-Gln66 E. coli IL-1β Ala117-Ser269 E. coli IL-2 Ala21-Thr153 E. coli IL-4 His25-Ser153 E. coli IL-6 Pro29-Met212 E. coli IL-8 Ser28-Ser99 E. coli IL-10 Ser19-Asn178 sf21 insect cells IL-12p70 IL-12p40 (Ile23-Ser328) sf21 insect cells IL-12p35 (Arg23-Ser219) IL-13 Gly21-Asn132 E. coli TNFα Val11-Leu233 E. coli

TABLE 66 Panel 2 Calibrator Sequence Expression System GM-CSF Ala18-Glu144 E. coli IL-1α Ser113-Ala271 E. coli IL-5 Ile20-Ser134 sf21 insect cells IL-7 Asp26-His177 E. coli IL-12/IL-23 p40 Ile23-Ser328 sf21 insect cells IL-15 Asn49-Ser162 E. coli IL-16 Pro2-Ser130 E. coli IL-17A Ile-20-Ala155 E. coli TNFβ Leu35-Leu205 E. coli VEGF Ala21-Arg191 sf21 insect cells

TABLE 67 Panel 3 Calibrator Sequence Expression System Eotaxin Gly24-Pro97 E. coli MIP-1β Ala24-Asn92 E. coli Eotaxin-3 Thr24-Leu94 E. coli TARC Ala24-Ser94 E. coli IP-10 Val22-Pro98 E. coli MIP-1α Ala27-Ala92 E. coli IL-8 Ser28-Ser99 E. coli MCP-1 Gln24-Thr99 E. coli MDC Gly25-Gln93 E. coli MCP-4 Gln24-Thr98 E. coli

TABLE 68 Panel 4 Calibrator Sequence Expression System IFNγ Glu23-Cys156 E. coli IL-2 Ala21-Gln155 E. coli IL-4 Cys25-Ser147 E. coli IL-1β Val117-Ser268 E. coli IL-5 Met20-Val132 E. coli IL-6 Phe25-Thr211 E. coli KC/GRO Ala25-Lys96 E. coli IL-10 Ser19-Asn178 E. coli IL-13 Thr19-His131 E. coli TNFα Leu80-Leu235 E. coli

TABLE 69 Panel 5 Calibrator Sequence Expression System IFNγ His23-Cys155 E. coli IL-1β Val118-Ser269 E. coli IL-2 Ala21-Gln169 E. coli IL-4 His23-Ser140 E. coli IL-5 Met21-Gly133 sf21 insect cells IL-6 Phe25-Thr211 E. coli KC/GRO Arg20-Lys96 E. coli IL-10 Ser19-Ser178 E. coli IL-12p70 Met23-Ser335 (mouse sf21 insect cells IL-12p40) & Arg23-Ala215 (mouse IL-12p35) TNFα Leu80-Leu235 E. coli

The following antibodies, capture and detection, were used in each panel as follows:

TABLE 70 Panel 1 Source Species Analyte MSD Capture Antibody MSD Detection Antibody IFNγ Mouse Monoclonal Mouse Monoclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-2 Mouse Monoclonal Mouse Monoclonal IL-4 Mouse Monoclonal Mouse Monoclonal IL-6 Mouse Monoclonal Goat Polyclonal IL-8 Mouse Monoclonal Goat Polyclonal IL-10 Mouse Monoclonal Mouse Monoclonal IL-12p70 Mouse Monoclonal Mouse Monoclonal IL-13 Rat Monoclonal Mouse Monoclonal TNFα Mouse Monoclonal Goat Polyclonal

TABLE 71 Panel 2 Source Species Analyte MSD Capture Antibody MSD Detection Antibody GM-CSF Mouse Monoclonal Rat Monoclonal IL-1α Mouse Monoclonal Goat Polyclonal IL-5 Mouse Monoclonal Mouse Monoclonal IL-7 Mouse Monoclonal Goat Polyclonal IL-12/IL-23 p40 Mouse Monoclonal Mouse Monoclonal IL-15 Mouse Monoclonal Mouse Monoclonal IL-16 Mouse Monoclonal Goat Polyclonal IL-17A Mouse Monoclonal Goat Polyclonal TNFβ Mouse Monoclonal Mouse Monoclonal VEGF Mouse Monoclonal Mouse Monoclonal

TABLE 72 Panel 3 Source Species Analyte MSD Capture Antibody MSD Detection Antibody Eotaxin Mouse Monoclonal Mouse Monoclonal MIP-1β Mouse Monoclonal Mouse Monoclonal Eotaxin-3 Mouse Monoclonal Mouse Monoclonal TARC Mouse Monoclonal Mouse Monoclonal IP-10 Mouse Monoclonal Mouse Monoclonal MIP-1α Mouse Monoclonal Mouse Monoclonal IL-8 Mouse Monoclonal Goat Polyclonal MCP-1 Mouse Monoclonal Mouse Monoclonal MDC Mouse Monoclonal Mouse Monoclonal MCP-4 Mouse Monoclonal Mouse Monoclonal

TABLE 73 Panel 4 Source Species Analyte MSD Capture Antibody MSD Detection Antibody IFNγ Mouse Monoclonal Goat Polyclonal IL-2 Mouse Monoclonal Goat Polyclonal IL-4 Mouse Monoclonal Goat Polyclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-5 Rat Monoclonal Rat Monoclonal IL-6 Mouse Monoclonal Goat Polyclonal KC/GRO Mouse Monoclonal Goat Polyclonal IL-10 Mouse Monoclonal Goat Polyclonal IL-15 Mouse Monoclonal Goat Polyclonal TNFα Hamster Monoclonal Goat Polyclonal

TABLE 74 Panel 5 Source Species Analyte MSD Capture Antibody MSD Detection Antibody IFNγ Rat Monoclonal Rat Monoclonal IL-1β Mouse Monoclonal Goat Polyclonal IL-2 Rat Monoclonal Rat Monoclonal IL-4 Rat Monoclonal Rat Monoclonal IL-5 Rat Monoclonal Rat Monoclonal IL-6 Rat Monoclonal Goat Polyclonal KC/GRO Rat Monoclonal Goat Polyclonal IL-10 Rat Monoclonal Goat Polyclonal IL-12p70 Rat Monoclonal Rat Monoclonal TNFα Hamster Monoclonal Goat Polyclonal

Various publications and test methods are cited herein, the disclosures of which are incorporated herein by reference in their entireties, In cases where the present specification and a document incorporated by reference and/or referred to herein include conflicting disclosure, and/or inconsistent use of terminology, and/or the incorporated/referenced documents use or define terms differently than they are used or defined in the present specification, the present specification shall control.

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1.-20. (canceled)
 21. A method of manufacturing a lot of kits used in the analysis of a cytokine panel, wherein said kit comprises qualified detection and capture antibodies specific for one of the following sets of analytes: (i) human analytes: IFN-gamma, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha; (ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A; (iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4; (iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or (v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha; said method comprising the steps of subjecting a subset of kits in said lot to plate coating uniformity testing and passing said lot based on results of said uniformity testing.
 22. The method of claim 21 wherein said lot meets a specification selected from the group consisting of: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.
 23. The method of claim 21 wherein said lot meets the following specifications: (a) average intraplate CV of ≦10%; (b) maximum intraplate CV of ≦13%; (c) average uniformity metric of ≦25%; (d) maximum uniformity metric of ≦37%; (e) CV of intraplate averages of ≦18%; (f) lower signal boundary of >1500; and (g) upper signal boundary of <10⁶.
 24. A method of manufacturing a kit used in the analysis of a cytokine panel, wherein said kit comprises qualified detection and capture antibodies specific for one of the following sets of analytes: (i) human analytes: IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNFalpha; (ii) human analytes: GM-CSF, IL-1alpha, IL-5, IL-7, IL-12/IL-23 p40, IL-15, IL-16, IL-17A, TNF-beta, and VEGF-A; (iii) human analytes: Eotaxin, MIP-1 alpha, Eotaxin-3, TARC, IP-10, MIP-1 beta, IL-8, MCP-1, MDC, and MCP-4; (iv) rat analytes: IFN-gamma, IL-2, IL-4, IL-1 beta, IL-5, IL-6, KC/GRO, IL-10, IL-13, and TNF-alpha; or (v) mouse analytes: IFN-gamma, IL-1-beta, IL-2, IL-4, IL-5, IL-6, KC/GRO, IL-10, IL-12p70, and TNF-alpha; said method comprising the steps of: (a) subjecting a preliminary set of detection antibodies specific for said mouse analytes to CIEF, DLS, and Experion; (b) selecting qualified detection antibodies from said preliminary set of detection antibodies based on said CIEF, DLS, and Experion testing; (c) subjecting a preliminary set of capture antibodies specific for said mouse analytes to CIEF, DLS, and Experion; and (b) selecting qualified capture antibodies from said preliminary set of capture antibodies based on said CIEF, DLS, and Experion testing.
 25. The method of claim 24, wherein said method further comprises subjecting said preliminary set of detection antibodies to an additional analytical method selected from the group consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof.
 26. The method of claim 24, wherein said method further comprises subjecting said preliminary set of detection antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS.
 27. The method of claim 24, wherein said method further comprises subjecting said preliminary set of capture antibodies to an additional analytical method selected from the group consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinations thereof.
 28. The method of claim 24, wherein said method further comprises subjecting said preliminary set of capture antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS.
 29. The method of claim 24 wherein said method further comprises subjecting each of said preliminary set of detection and capture antibodies to an additional analytical method consisting of denaturing SDS-PAGE, non-denaturing SDS-PAGE, and SEC-MALS. 